Lipid-associated molecules

ABSTRACT

The invention provides human lipid-associated molecules (LIPAM) and polynucleotides which identify and encode LIPAM. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating, or preventing disorders associated with aberrant expression of LIPAM.

TECHNICAL FIELD

[0001] This invention relates to nucleic acid and amino acid sequencesof lipid-associated molecules and to the use of these sequences in thediagnosis, treatment, and prevention of cancers, neurological,autoimmune/inflammatory, gastrointestinal, and cardiovascular disorders,and disorders of lipid metabolism, and in the assessment of the effectsof exogenous compounds on the expression of nucleic acid and amino acidsequences of lipid-associated molecules.

BACKGROUND OF THE INVENTION

[0002] Lipids are water-insoluble, oily or greasy substances that aresoluble in nonpolar solvents such as chloroform or ether. Neutral fats(triacylglycerols) serve as major fuels and energy stores. Fatty acidsare long-chain organic acids with a single carboxyl group and a longnon-polar hydrocarbon tail. Long-chain fatty acids are essentialcomponents of glycolipids, phospholipids, and cholesterol, which arebuilding blocks for biological membranes, and of triglycerides, whichare biological fuel molecules. Lipids, such as phospholipids,sphingolipids, glycolipids, and cholesterol, are key structuralcomponents of cell membranes. Lipids and proteins are associated in avariety of ways. Glycolipids form vesicles that carry proteins withincells and cell membranes. Interactions between lipids and proteinsfunction in targeting proteins and glycolipids involved in a variety ofprocesses, such as cell signaling and cell proliferation, to specificmembrane and intracellular locations. Various proteins are associatedwith the biosynthesis, transport, and uptake of lipids. In addition, keyproteins involved in signal transduction and protein targeting havelipid-derived groups added to them post-translationally (Stryer, L.(1995) Biochemistry, W. H. Freeman and Co., New York N.Y., pp. 264-267,934; Lehninger, A. (1982) Principles of Biochemistry, Worth Publishers,Inc. New York N.Y.; and ExPASy “Biochemical Pathways” index ofBoehringer Mannheim World Wide Web site,“http://www.expasy.ch/cgi-bin/search-biochem-index”.)

[0003] Phospholipids

[0004] A major class of phospholipids are the phosphoglycerides, whichare composed of a glycerol backbone, two fatty acid chains, and aphosphorylated alcohol. Phosphoglycerides are components of cellmembranes. Principal phosphoglycerides are phosphatidyl choline,phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol,and diphosphatidyl glycerol. Many enzymes involved in phosphoglyceridesynthesis are associated with membranes (Meyers, R. A. (1995) MolecularBiology and Biotechnology, VCH Publishers Inc., New York N.Y., pp.494-501). Phosphatidate is converted to CDP-diacylglycerol by the enzymephosphatidate cytidylyltransferase (ExPASy ENZYME EC 2.7.7.41). Transferof the diacylglycerol group from CDP-diacylglycerol to serine to yieldphosphatidyl serine, or to inositol to yield phosphatidyl inositol, iscatalyzed by the enzymes CDP-diacylglycerol-serineO-phosphatidyltransferase and CDP-diacylglycerol-inositol3-phosphatidyltransferase, respectively (ExPASy ENZYME EC 2.7.8.8;ExPASy ENZYME EC 2.7.8.11). The enzyme phosphatidyl serine decarboxylasecatalyzes the conversion of phosphatidyl serine to phosphatidylethanolamine, using a pyruvate cofactor (Voelker, D. R. (1997) Biochim.Biophys. Acta 1348:236-244). Phosphatidyl choline is formed usingdiet-derived choline by the reaction of CDP-choline with1,2-diacylglycerol, catalyzed by diacylglycerolcholinephosphotransferase (ExPASy ENZYME 2.7.8.2).

[0005] Other phosphoglycerides have been shown to be involved in thevesicle trafficking process. Phosphatidylinositol transfer protein(PITP) is a ubiquitous cytosolic protein, thought to be involved intransport of phospholipids from their site of synthesis in theendoplasmic reticulum and Golgi to other cell membranes. More recently,PITP has been shown to be an essential component of thepolyphosphoinositide synthesis machinery and is hence required forproper signaling by epidermal growth factor and f-Met-Leu-Phe, as wellas for exocytosis. The role of PITI in polyphosphoinositide synthesismay also explain its involvement in intracellular vesicular traffic(Liscovitch, M. et al. (1995) Cell 81:659-662).

[0006] The copines are phospholipid-binding proteins believed tofunction in membrane trafficking. Copines promote lipid vesicleaggregation. They contain a C2 domain associated with membrane activityand an annexin-type domain that mediates interactions between integraland extracellular proteins and is associated with calcium binding andregulation (Creutz, C. E. (1998) J. Biol. Chem. 273:1393-1402). OtherC2-containing proteins include the synaptotagmins, a family of proteinsinvolved in vesicular trafficking. Synaptotagmin concentrations incerebrospinal fluid have been found to be reduced in early-onsetAlzheimer's disease (Gottfries, C. G. et al. (1998) J. Neural Transm.105:773-786).

[0007] The phosphatidylinositol-transfer protein Sec14, which catalysesexchange of phosphatidylinositol and phosphatidylcholine betweenmembrane bilayers in vitro, is essential for vesicle budding from theGolgi complex. Sec14 includes a carboxy-terminal domain that forms ahydrophobic pocket which represents the phospholipid-binding domain(Sha, B. et al. (1998) Nature 391:506-510). Sec14 is a member of thecellular retinaldehyde-binding protein (CRAL)/Triple function domain(TRIO) family (InterPro Entry IPR001251, http://www.ebi.ac.uk/interpro).

[0008] Sphingolipids

[0009] Sphingolipids are an important class of membrane lipids thatcontain sphingosine, a long chain amino alcohol. They are composed ofone long-chain fatty acid, one polar head alcohol, and sphingosine orsphingosine derivatives. The three classes of sphingolipids aresphingomyelins, cerebrosides, and gangliosides. Sphingomyelins, whichcontain phosphocholine or phosphoethanolamine as their head group, areabundant in the myelin sheath surrounding nerve cells.Galactocerebrosides, which contain a glucose or galactose head group,are characteristic of the brain. Other cerebrosides are found innon-neural tissues. Gangliosides, whose head groups contain multiplesugar units, are abundant in the brain, but are also found in non-neuraltissues.

[0010] Glycolipids

[0011] Glycolipids are also important components of the plasma membranesof animal cells. The most simple glycolipid is cerebroside whichcomprises only a single glucose or galactose sugar residue in additionto the lipid component. Gangliosides are glycosphingolipid plasmamembrane components that are abundant in the nervous systems ofvertebrates. Gangliosides are the most complex glycolipids and compriseceramide (acylated sphingosine) attached to an oligosaccharide moietycontaining at least one acidic sugar residue (sialic acid), namelyN-acetylneuraminate or N-glycolylneuraminate. The sugar residues areadded sequentially to ceramide via UDP-glucose, UDP-galactose,N-acetylgalactosamine, and CMP-N-acetylneuraminate donors. Over 15gangliosides have been identified with G_(M1) and G_(M2) being the bestcharacterized (Stryer, L. (1988) Biochemistry, W. H. Freeman and Co.,Inc., New York, pp. 552-554).

[0012] Gangliosides are thought to play important roles in cell surfaceinteractions, cell differentiation, neuritogenesis, the triggering andmodulation of transmembrane signaling, mediatiosynaptic function, neuralrepair, neurite outgrowth, and neuronal death (Hasegawa, T. et al.(2000) J. Biol. Chem. 275:8007-8015). While the presence of gangliosidesin the plasma membrane is important for orchestrating these events, thesubsequent removal of carbohydrate groups (desialylation) by sialidasesalso appears to be important for regulating neuronal differentiation.

[0013] Specific soluble N-ethylmaleimide-sensitive factor attachmentprotein (SNAP) receptor (SNARE) proteins are required for differentmembrane transport steps. The SNARE protein Vti1a has been colocalizedwith Golgi markers while Vti1b has been colocalized with Golgi and thetrans-Golgi network of endosomal markers in fibroblast cell lines. Abrain-specific splice variant of Vti1a is enriched in small synapticvesicles and clathrin-coated vesicles isolated from nerve terminals.Vti1a-beta and synaptobrevin are integral parts of synaptic vesiclesthroughout their life cycle. Vti1a-beta functions in a SNARE complexduring recycling or biogenesis of synaptic vesicles (Antonin, W. et al.(2000) J. Neurosci. 20:5724-5732).

[0014] Sialidases catalyze the first step in glycosphingolipiddegradation, removing carbohydrate moieties from gangliosides. Theseenzymes are present in the cytosol, lysosomal matrix, lysosomalmembrane, and plasma membrane (Hasegawa, T. et al. (2000) J. Biol. Chem.275:8007-8015). Hallmark features of sialidases include a transmembranedomain, an Arg-Ile-Pro domain, and three Asp-box sequences (Wada, T.(1999) Biochem. Biophys.Res. Commun. 261:21-27).

[0015] During normal neuronal development, pyramidal neurons of thecerebral cortex participate in a single burst of dendritic sproutingimmediately following nerve cell migration to the cortical mantle. Cellsundergoing dendritogenesis are characterized by increased expression ofG_(M2) ganglioside which decreases following dentritic maturation.Evidence suggests that no new primary dendrites are initiated followingthe initial burst.

[0016] Cholesterol

[0017] Cholesterol, composed of four fused hydrocarbon rings with analcohol at one end, moderates the fluidity of membranes in which it isincorporated. In addition, cholesterol is used in the synthesis ofsteroid hormones such as cortisol, progesterone, estrogen, andtestosterone. Bile salts derived from cholesterol facilitate thedigestion of lipids. Cholesterol in the skin forms a barrier thatprevents excess water evaporation from the body. Farnesyl andgeranylgeranyl groups, which are derived from cholesterol biosynthesisintermediates, are post-translationally added to signal transductionproteins such as Ras and protein-targeting proteins such as Rab. Thesemodifications are important for the activities of these proteins(Guyton, A. C. (1991) Textbook of Medical Physiology, W. B. SaundersCompany, Philadelphia Pa., pp. 760-763; Stryer, supra, pp. 279-280,691-702, 934).

[0018] Mammals obtain cholesterol derived from both de novo biosynthesisand the diet. The liver is the major site of cholesterol biosynthesis inmammals. Biosynthesis is accomplished via a series of enzymatic stepsknown as the mevalonate pathway. The rate-limiting step is theconversion of hydroxymethylglutaryl-Coenzyme A (HMG-CoA) to mevalonateby HMG-CoA reductase. The drug lovastatin, a potent inhibitor of HMG-CoAreductase, is given to patients to reduce their serum cholesterollevels. Cholesterol derived from de novo biosynthesis or from the dietis transported in the body fluids in the form of lipoprotein particles.These particles also transport triacylglycerols. The particles consistof a core of hydrophobic lipids surrounded by a shell of polar lipidsand apolipoproteins. The protein components serve in the solubilizationof hydrophobic lipids and also contain cell-targeting signals.Lipoproteins include chylomicrons, chylomicron remnants,very-low-density lipoproteins (VLDL), intermediate-density lipoproteins(IDL), low-density lipoproteins (LDL), and high-density lipoproteins(HDL) (Meyers, supra; Stryer, supra, pp. 691-702). There is a stronginverse correlation between the levels of plasma HDL and risk ofpremature coronary heart disease. ApoL is an HDL apolipoproteinexpressed in the pancreas (Duchateau, P. N. et al. (1997) J. Biol. Chem.272:25576-25582).

[0019] Most cells outside the liver and intestine take up cholesterolfrom the blood rather than synthesize it themselves. Cell surface LDLreceptors bind LDL particles which are then internalized by endocytosis(Meyers, supra). Absence of the LDL receptor, the cause of the diseasefamilial hypercholesterolemia, leads to increased plasma cholesterollevels and ultimately to atherosclerosis (Stryer, supra, pp. 691-702).

[0020] Proteins involved in cholesterol uptake and biosynthesis aretightly regulated in response to cellular cholesterol levels. The sterolregulatory element binding protein (SREBP) is a sterol-responsivetranscription factor. Under normal cholesterol conditions, SREBP residesin the endoplasmic reticulum membrane. When cholesterol levels are low,a regulated cleavage of SREBP occurs which releases the extracellulardomain of the protein. This cleaved domain is then transported to thenucleus where it activates the transcription of the LDL receptor gene,and genes encoding enzymes of cholesterol-synthesis, by binding thesterol regulatory element (SRE) upstream of the genes (Yang, J. et al.(1995) J. Biol. Chem. 270:12152-12161). Regulation of cholesterol uptakeand biosynthesis also occurs via the oxysterol-binding protein (OSBP).Oxysterols are oxidation products formed during the catabolism ofcholesterol, and are involved in regulation of steroid biosynthesis.OSBP is a high-affinity intracellular receptor for a variety ofoxysterols that down-regulate cholesterol synthesis and stimulatecholesterol esterification (Lagace, T. A. et al. (1997) Biochem. J.326:205-213).

[0021] Supernatant protein factor (SPF), which stimulates squaleneepoxidation and conversion of squalene to lanosterol, is a cytosolicsqualene transfer protein that enhances cholesterol biosynthesis.Squalene epoxidase, a membrane-associated enzyme that converts squaleneto squalene 2,3-oxide, plays an important role in the maintenance ofcholesterol homeostasis. SPF belongs to a family of cytosoliclipid-binding/transfer proteins such as alpha-tocopherol transferprotein, cellular retinal binding protein, yeast phosphatidylinositoltransfer protein (Sec14p), and squid retinal binding protein (Shibata,N. et al. (2001) Proc. Natl. Acad. Sci. USA 98:2244-2249).

[0022] Lipid Metabolism Enzymes

[0023] Long-chain fatty acids are also substrates for eicosanoidproduction, and are important in the functional modification of certaincomplex carbohydrates and proteins. 16-carbon and 18-carbon fatty acidsare the most common. Fatty acid synthesis occurs in the cytoplasm. Inthe first step, acetyl-Coenzyme A (CoA) carboxylase (ACC) synthesizesmalonyl-CoA from acetyl-CoA and bicarbonate. The enzymes which catalyzethe remaining reactions are covalently linked into a single polypeptidechain, referred to as the multifunctional enzyme fatty acid synthase(FAS). FAS catalyzes the synthesis of palmitate from acetyl-CoA andmalonyl-CoA. FAS contains acetyl transferase, malonyl transferase,β-ketoacetyl synthase, acyl carrier protein, β-ketoacyl reductase,dehydratase, enoyl reductase, and thioesterase activities. The finalproduct of the FAS reaction is the 16-carbon fatty acid palmitate.Further elongation, as well as unsaturation, of palmitate by accessoryenzymes of the ER produces the variety of long chain fatty acidsrequired by the individual cell. These enzymes include a NADH-cytochromeb₅ reductase, cytochrome b₅, and a desaturase.

[0024] Within cells, fatty acids are transported by cytoplasmic fattyacid binding proteins (Online Mendelian Inheritance in Man (OMIM)*134650 Fatty Acid-Binding Protein 1, Liver; FABP1). Diazepam bindinginhibitor (DBI), also known as endozepine and acyl CoA-binding protein,is an endogenous γ-aminobutyric acid (GABA) receptor ligand which isthought to down-regulate the effects of GABA. DBI binds medium- andlong-chain acyl-CoA esters with very high affinity and may function asan intracellular carrier of acyl-CoA esters (OMIM *125950 DiazepamBinding Inhibitor; DBI; PROSITE PDOC00686 Acyl-CoA-binding proteinsignature).

[0025] Fat stored in liver and adipose triglycerides may be released byhydrolysis and transported in the blood. Free fatty acids aretransported in the blood by albumin. Triacylglycerols, also known astriglycerides and neutral fats, are major energy stores in animals.Triacylglycerols are esters of glycerol with three fatty acid chains.Glycerol-3-phosphate is produced from dihydroxyacetone phosphate by theenzyme glycerol phosphate dehydrogenase or from glycerol by glycerolkinase. Fatty acid-CoAs are produced from fatty acids by fatty acyl-CoAsynthetases. Glyercol-3-phosphate is acylated with two fatty acyl-CoAsby the enzyme glycerol phosphate acyltransferase to give phosphatidate.Phosphatidate phosphatase converts phosphatidate to diacylglycerol,which is subsequently acylated to a triacylglyercol by the enzymediglyceride acyltransferase. Phosphatidate phosphatase and diglycerideacyltransferase form a triacylglyerol synthetase complex bound to the ERmembrane.

[0026] Mitochondrial and peroxisomal beta-oxidation enzymes degradesaturated and unsaturated fatty acids by sequential removal oftwo-carbon units from CoA-activated fatty acids. The main beta-oxidationpathway degrades both saturated and unsaturated fatty acids while theauxiliary pathway performs additional steps required for the degradationof unsaturated fatty acids. The pathways of mitochondrial andperoxisomal beta-oxidation use similar enzymes, but have differentsubstrate specificities and functions. Mitochondria oxidize short-,medium-, and long-chain fatty acids to produce energy for cells.Mitochondrial beta-oxidation is a major energy source for cardiac andskeletal muscle. In liver, it provides ketone bodies to the peripheralcirculation when glucose levels are low as in starvation, enduranceexercise, and diabetes (Eaton, S. et al. (1996) Biochem. J.320:345-357). Peroxisomes oxidize medium-, long-, and very-long-chainfatty acids, dicarboxylic fatty acids, branched fatty acids,prostaglandins, xenobiotics, and bile acid intermediates. The chiefroles of peroxisomal beta-oxidation are to shorten toxic lipophiliccarboxylic acids to facilitate their excretion and to shortenvery-long-chain fatty acids prior to mitochondrial beta-oxidation(Mannaerts, G. P. and P. P. Van Veldhoven (1993) Biochimie 75:147-158).Enzymes involved in beta-oxidation include acyl CoA synthetase,carnitine acyltransferase, acyl CoA dehydrogenases, enoyl CoAhydratases, L-3-hydroxyacyl CoA dehydrogenase, β-ketothiolase,2,4-dienoyl CoA reductase, and isomerase.

[0027] Three classes of lipid metabolism enzymes are discussed infurther detail. The three classes are lipases, phospholipases andlipoxygenases.

[0028] Lipases

[0029] Triglycerides are hydrolyzed to fatty acids and glycerol bylipases. Adipocytes contain lipases that break down storedtriacylglycerols, releasing fatty acids for export to other tissueswhere they are required as fuel. Lipases are widely distributed inanimals, plants, and prokaryotes. Triglyceride lipases (ExPASy ENZY EC3.1.1.3), also known as triacylglycerol lipases and tributyrases,hydrolyze the ester bond of triglycerides. In higher vertebrates thereare at least three tissue-specific isozymes including gastric, hepatic,and pancreatic lipases. These three types of lipases are structurallyclosely related to each other as well as to lipoprotein lipase. The mostconserved region in gastric, hepatic, and pancreatic lipases is centeredaround a serine residue which is also present in lipases of prokaryoticorigin. Mutation in the serine residue renders the enzymes inactive.Gastric, hepatic, and pancreatic lipases hydrolyze lipoproteintriglycerides and phospholipids. Gastric lipases in the intestine aid inthe digestion and absorption of dietary fats. Hepatic lipases are boundto and act at the endothelial surfaces of hepatic tissues. Hepaticlipases also play a major role in the regulation of plasma lipids.Pancreatic lipase requires a small protein cofactor, colipase, forefficient dietary lipid hydrolysis. Colipase binds to the C-terminal,non-catalytic domain of lipase, thereby stabilizing an activeconformation and considerably increasing the overall hydrophobic bindingsite. Deficiencies of these enzymes have been identified in man, and allare associated with pathologic levels of circulating lipoproteinparticles (Gargouri, Y. et al. (1989) Biochim. Biophys. Acta1006:255-271; Connelly, P. W. (1999) Clin. Chim. Acta 286:243-255; vanTilbeurgh, H. et al. (1999) Biochim. Biophys. Acta 1441:173-184).

[0030] Lipoprotein lipases (ExPASy ENZYME EC 3.1.1.34), also known asclearing factor lipases, diglyceride lipases, or diacylglycerol lipases,hydrolyze triglycerides and phospholipids present in circulating plasmalipoproteins, including chylomicrons, very low and intermediate densitylipoproteins and high-density lipoproteins (HDL). Together withpancreatic and hepatic lipases, lipoprotein lipases (LPL) share a highdegree of primary sequence homology. Both lipoprotein lipases andhepatic lipases are anchored to the capillary endothelium viaglycosaminoglycans and can be released by intravenous administration ofheparin. LPLs are primarily synthesized by adipocytes, muscle cells, andmacrophages. Catalytic activities of LPLs are activated byapolipoprotein C-II and are inhibited by high ionic strength conditionssuch as 1 M NaCl. LPL deficiencies in humans contribute to metabolicdiseases such as hypertriglyceridemia, HDL2 deficiency, and obesity(Jackson, R. L. (1983) in The Enzymes (Boyer, P. D., ed.) Vol. XVI, pp.141-186, Academic Press, New York N.Y.; Eckel, R. H. (1989) New Engl. J.Med. 320:1060-1068).

[0031] Phospholipases

[0032] Phospholipases, a group of enzymes that catalyze the hydrolysisof membrane phospholipids, are classified according to the bond cleavedin a phospholipid. They are classified into PLA1, PLA2, PLB, PLC, andPLD families. Phospholipases are involved in many inflammatory reactionsby making arachidonate available for eicosanoid biosynthesis. Morespecifically, arachidonic acid is processed into bioactive lipidmediators of inflammation such as lyso-platelet-activating factor andeicosanoids. The synthesis of arachidonic acid from membranephospholipids is the rate-limiting step in the biosynthesis of the fourmajor classes of eicosanoids (prostaglandins, prostacyclins,thromboxanes and leukotrienes), whcih are 20-carbon molecules derivedfrom fatty acids. Eicosanoids are signaling molecules which have rolesin pain, fever, and inflammation. The precursor of all eicosanoids isarachidonate, which is generated from phospholipids by phospholipase A₂and from diacylglycerols by diacylglycerol lipase. Leukotrienes areproduced from arachidonate by the action of lipoxygenases (Kaiser, E. etal. (1990) Clin. Biochem. 23:349-370). Furthermore, leukotriene-B4 isknown to function in a feedback loop which further increases PLA2activity (Wijkander, J. et al. (1995) J. Biol. Chem. 270:26543-26549).

[0033] The secretory phospholipase A₂ (PLA2) superfamily comprises anumber of heterogeneous enzymes whose common feature is to hydrolyze thesn-2 fatty acid acyl ester bond of phosphoglycerides. Hydrolysis of theglycerophospholipids releases free fatty acids and lysophospholipids.PLA2 activity generates precursors for the biosynthesis of biologicallyactive lipids, hydroxy fatty acids, and platelet-activating factor.PLA2s were first described as components of snake venoms, and were latercharacterized in numerous species. PLA2s have traditionally beenclassified into several major groups and subgroups based on their aminoacid sequences, divalent cation requirements, and location of disulfidebonds. The PLA2s of Groups I, II, and III consist of low molecularweight, secreted, Ca²⁺-dependent proteins. Group IV PLA2s are primarily85-kDa, Ca²⁺-dependent cytosolic phospholipases. Finally, a number ofCa²⁺-independent PLA2s have been described, which comprise Group V(Davidson, F. F. and E. A. Dennis (1990) J. Mol. Evol. 31:228-238; andDennis, E. F. (1994) J. Biol Chem. 269:13057-13060).

[0034] The first PLA2s to be extensively characterized were the Group I,II, and III PLA2s found in snake and bee venoms. These venom PLA2s sharemany features with mammalian PLA2s including a common catalyticmechanism, the same Ca²⁺ requirement, and conserved primary and tertiarystructures. In addition to their role in the digestion of prey, thevenom PLA2s display neurotoxic, myotoxic, anticoagulant, andproinflammatory effects in mammalian tissues. This diversity ofpathophysiological effects is due to the presence of specific, highaffinity receptors for these enzymes on various cells and tissues(Lambeau, G. et al. (1995) J. Biol. Chem. 270:5534-5540).

[0035] PLA2s from Groups I, IIA, IIC, and V have been described inmammalian and avian cells, and were originally characterized by tissuedistribution, although the distinction is no longer absolute. Thus,Group I PLA2s were found in the pancreas, Group IIA and IIC were derivedfrom inflammation-associated tissues (e.g., the synovium), and Group Vwere from cardiac tissue. The pancreatic PLA2s function in the digestionof dietary lipids and have been proposed to play a role in cellproliferation, smooth muscle contraction, and acute lung injury. TheGroup II inflammatory PLA2s are potent mediators of inflammatoryprocesses and are highly expressed in serum and synovial fluids ofpatients with inflammatory disorders. These Group II PLA2s are found inmost human cell types assayed and are expressed in diverse pathologicalprocesses such as septic shock, intestinal cancers, rheumatoidarthritis, and epidermal hyperplasia. A Group V PLA2 has been clonedfrom brain tissue and is strongly expressed in heart tissue. A humanPLA2 was recently cloned from fetal lung, and based on its structuralproperties, appears to be the first member of a new group of mammalianPLA2s, referred to as Group X. Other PLA2s have been cloned from varioushuman tissues and cell lines, suggesting a large diversity of PLA2s(Chen, J. et al. (1994) J. Biol. Chem. 269:2365-2368; Kennedy, B. P. etal. (1995) J. Biol. Chem. 270: 22378-22385; Komada, M. et al. (1990)Biochem. Biophys. Res. Commun. 168:1059-1065; Cupillard, L. et al.(1997) J. Biol. Chem. 272:15745-15752; and Nalefski, E. A. et al. (1994)J. Biol. Chem. 269:18239-18249).

[0036] Phospholipases B (PLB) (ExPASy ENZYME EC 3.1.1.5), also known aslysophospholipase, lecithinase B, or lysolecithinase are widelydistributed enzymes that metabolize intracellular lipids, and occur innumerous isoforms. Small isoforms, approximately 15-30 kD, function ashydrolases; large isoforms, those exceeding 60 kD, function both ashydrolases and transacylases. A particular substrate for PLBs,lysophosphatidylcholine, causes lysis of cell membranes when it isformed or imported into a cell. PLBs are regulated by lipid factorsincluding acylcarnitine, arachidonic acid, and phosphatidic acid. Theselipid factors are signaling molecules important in numerous pathways,including the inflammatory response (Anderson, R. et al. (1994) Toxicol.Appl. Pharmacol. 125:176-183; Selle, H. et al. (1993); Eur. J. Biochem.212:411-416).

[0037] Phospholipase C (PLC) (ExPASy ENZYME EC 3.1.4.10) plays animportant role in transmembrane signal transduction. Many extracellularsignaling molecules including hormones, growth factors,neurotransmitters, and immunoglobulins bind to their respective cellsurface receptors and activate PLCs. The role of an activated PLC is tocatalyze the hydrolysis of phosphatidyl-inositol-4,5-bisphosphate(PIP2), a minor component of the plasma membrane, to producediacylglycerol and inositol 1,4,5-trisphosphate (IP3). In theirrespective biochemical pathways, IP3 and diacylglycerol serve as secondmessengers and trigger a series of intracellular responses. IP3 inducesthe release of Ca²⁺ from internal cellular storage, and diacylglycerolactivates protein kinase C (PKC). Both pathways are part oftransmembrane signal transduction mechanisms which regulate cellularprocesses which include secretion, neural activity, metabolism, andproliferation.

[0038] Several distinct isoforms of PLC have been identified and arecategorized as PLC-beta, PLC-gamma, and PLC-delta. Subtypes aredesignated by adding Arabic numbers after the Greek letters, eg.PLC-β-1. PLCs have a molecular mass of 62-68 kDa, and their amino acidsequences show two regions of significant similarity. The first region,designated X, has about 170 amino acids, and the second, or Y region,contains about 260 amino acids.

[0039] The catalytic activities of the three isoforms of PLC aredependent upon Ca²⁺. It has been suggested that the binding sites forCa²⁺ in the PLCs are located in the Y-region, one of two conservedregions. The hydrolysis of common inositol-containing phospholipids,such as phosphatidylinositol (PI), phosphatidylinositol 4-monophosphate(PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2), by any of theisoforms yields cyclic and noncyclic inositol phosphates (Rhee, S. G.and Y. S. Bae (1997) J. Biol. Chem. 272:15045-15048).

[0040] All mammalian PLCs contain a pleckstrin homology (PH) domainwhich is about 100 amino acids in length and is composed of twoantiparallel beta sheets flanked by an amphipathic alpha helix. PHdomains target PLCs to the membrane surface by interacting with eitherthe beta/gamma subunits of G proteins or PIP2 (PROSITE PDOC50003).

[0041] Phospholipase D (PLD) (ExPASy ENZYME EC 3.1.4.4), also known aslecithinase D, lipophosphodiesterase II, and choline phosphatasecatalyzes the hydrolysis of phosphatidylcholine and other phospholipidsto generate phosphatidic acid. PLD plays an important role in membranevesicle trafficking, cytoskeletal dynamics, and transmembrane signaltransduction. In addition, the activation of PLD is involved in celldifferentiation and growth (reviewed in Liscovitch, M. (2000) Biochem.J. 345:401-415).

[0042] PLD is activated in mammalian cells in response to diversestimuli that include hormones, neurotransmitters, growth factors,cytokines, activators of protein kinase C, and agonist binding toG-protein-coupled receptors. At least two forms of mammalian PLD, PLD1and PLD2, have been identified. PLD1 is activated by protein kinase Calpha and by the small GTPases ARF and RhoA. (Houle, M. G. and S.Bourgoin (1999) Biochim. Biophys. Acta 1439:135-149). PLD2 can beselectively activated by unsaturated fatty acids such as oleate (Kim, J.H. (1999) FEBS Lett. 454:42-46).

[0043] Lipoxygenases

[0044] Lipoxygenases (ExPASy ENZYME EC 1.13.11.12) are non-hemeiron-containing enzymes that catalyze the dioxygenation of certainpolyunsaturated fatty acids such as lipoproteins. Lipoxygenases arefound widely in plants, fungi, and animals. Several differentlipoxygenase enzymes are known, each having a characteristic oxidationaction. In animals, there are specific lipoxygenases that catalyze thedioxygenation of arachidonic acid at the carbon-3, 5, 8, 11, 12, and 15positions. These enzymes are named after the position of arachidonicacid that they dioxygenate. Lipoxygenases have a single polypeptidechain with a molecular mass of ˜75-80 kDa in animals. The proteins havean N-terminal-barrel domain and a larger catalytic domain containing asingle atom of non-heme iron. Oxidation of the ferric enzyme to anactive form is required for catalysis (Yamamoto, S. (1992) Biochim.Biophys. Acta 1128:117-131; Brash, A. R. (1999) J. Biol. Chem.274:23679-23682). A variety of lipoxygenase inhibitors exist and areclassified into five major categories according to their mechanism ofinhibition. These include antioxidants, iron chelators, substrateanalogues, lipoxygenase-activating protein inhibitors, and, finally,epidermal growth factor-receptor inhibitors.

[0045] 3-Lipoxygenase, also known as e-LOX-3 or Aloxe3 has recently beencloned from murine epidermis. Aloxe3 resides on mouse chromosome 11, andthe deduced amino acid sequence for Aloxe3 is 54% identical to the12-lipoxygenase sequences (Kinzig, A. (1999) Genomics 58:158-164).

[0046] 5-Lipoxygenase (5-LOX, ExPASy ENZYME EC 1.13.11.34), also knownas arachidonate:oxygen 5-oxidoreductase, is found primarily in whiteblood cells, macrophages, and mast cells. 5-LOX converts arachidonicacid first to 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and then toleukotriene (LTA4 (5,6-oxido-7,9,11,14-eicosatetraenoic acid)).Subsequent conversion of leukotriene A4 by leukotriene A4 hydrolaseyields the potent neutrophil chemoattractant leukotriene B4.Alternatively, conjugation of LTA4 with glutathione by leukotriene C4synthase plus downstream metabolism leads to the cysteinyl leukotrienesthat influence airway reactivity and mucus secretion, especially inasthmatics. Most lipoxygenases require no other cofactors or proteinsfor activity. In contrast, the mammalian 5-LOX requires calcium and ATP,and is activated in the presence of a 5-LOX activating protein (FLAP).FLAP itself binds to arachidonic acid and supplies 5-LOX with substrate(Lewis, R. A. et al. (1990) New Engl. J. Med. 323:645-655). Theexpression levels of 5-LOX and FLAP are found to be increased in thelungs of patients with plexogenic (primary) pulmonary hypertension(Wright, L. et al. (1998) Am. J. Respir. Crit. Care Med. 157:219-229).

[0047] 12-Lipoxygenase (12-LOX, ExPASy ENZYME: EC 1.13.11.31) oxygenatesarachidonic acid to form 12-hydroperoxyeicosatetraenoic acid (12-HPETE).Mammalian 12-lipoxygenases are named after the prototypical tissues oftheir occurrence (hence, the leukocyte, platelet, or epidermal types).Platelet-type 12-LOX has been found to be the predominant isoform inepidermal skin specimens and epidermoid cells. Leukocyte 12-LOX wasfirst characterized extensively from porcine leukocytes and was found tohave a rather broad distribution in mammalian tissues by immunochemicalassays. Besides tissue distribution, the leukocyte 12-LOX isdistinguished from the platelet-type enzyme by its ability to form15-HPETE, in addition to 12-HPETE, from arachidonic acid substrate.Leukocyte 12-LOX is highly related to 15-lipoxgenase (15-LOX) in thatboth are dual specificity lipoxygenases, and they are about 85%identical in primary structure in higher mammals. Leukocyte 12-LOX isfound in tracheal epithelium, leukocytes, and macrophages (Conrad, D. J.(1999) Clin. Rev. Allergy Immunol. 17:71-89).

[0048] 15-Lipoxygenase (15-LOX; ExPASy ENZYME: EC 1.13.11.33) is foundin human reticulocytes, airway epithelium, and eosinophils. 15-LOX hasbeen detected in atherosclerotic lesions in mammals, specifically rabbitand man. The enzyme, in addition to its role in oxidative modificationof lipoproteins, is important in the inflammatory reaction inatherosclerotic lesions. 15-LOX has been shown to be induced in humanmonocytes by the cytokine IL-4, which is known to be implicated in theinflammatory process (Kuhn, H. and S. Borngraber (1999) Adv. Exp. Med.Biol. 447:5-28).

[0049] A variety of lipolytic enzymes with a GDSL-like motif as part ofthe active site have been identified. Members of this family include alipase/acylhydrolase, thermolabile hemolysin and rabbit phospholipase(AdRab-B)(Interpro entry IPR001087, http://www.sanger.ac.uk). A homologof AdRab-B is guinea pig intestinal phospholipase B, acalcium-independent phospholipase that contributes to lipid digestion asan ectoenzyme by sequentially hydrolyzing the acyl ester bonds ofglycerophospholipids. Phospholipase B also has a role in malereproduction (Delagebeaudeuf, C. et al. (1998) J. Biol. Chem.273:13407-13414).

[0050] Lipid-Associated Molecules and Disease

[0051] Lipids and their associated proteins have roles in human diseasesand disorders. Increased synthesis of long-chain fatty acids occurs inneoplasms including those of the breast, prostate, ovary, colon andendometrium.

[0052] In the arterial disease atherosclerosis, fatty lesions form onthe inside of the arterial wall. These lesions promote the loss ofarterial flexibility and the formation of blood clots (Guyton, supra).There is a strong inverse correlation between the levels of plasma HDLand risk of premature coronary heart disease. Absence of the LDLreceptor, the cause of familial hypercholesterolemia, leads to increasedplasma cholesterol levels and ultimately to atherosclerosis (Stryer,supra, pp. 691-702). Oxysterols are present in human atheroscleroticplaques and are believed to play an active role in plaque development(Brown, A. J. (1999) Atherosclerosis 142:1-28). Lipases, phospholipases,and lipoxygenases are thought to contribute to complex diseases, such asatherosclerosis, obesity, arthritis, asthma, and cancer, as well as tosingle gene defects, such as Wolman's disease and Type Ihyperlipoproteinemia.

[0053] Steatosis, or fatty liver, is characterized by the accumulationof triglycerides in the liver and may occur in association with avariety of conditions including alcoholism, diabetes, obesity, andprolonged parenteral nutrition. Steatosis may lead to fibrosis andcirrhosis of the liver.

[0054] Niemann-Pick diseases types A and B are caused by accumulation ofsphingomyelin (a sphingolipid) and other lipids in the central nervoussystem due to a defect in the enzyme sphingomyelinase, leading toneurodegeneration and lung disease. Niemann-Pick disease type C resultsfrom a defect in cholesterol transport, leading to the accumulation ofsphingomyelin and cholesterol in lysosomes and a secondary reduction insphingomyelinase activity. Neurological symptoms such as grand malseizures, ataxia, and loss of previously learned speech, manifest 1-2years after birth. A mutation in the NPC protein, which contains aputative cholesterol-sensing domain, was found in a mouse model ofNiemann-Pick disease type C (Fauci, supra, p. 2175; Loftus, S. K. et al.(1997) Science 277:232-235).

[0055] Tay-Sachs disease is an autosomal recessive, progressiveneurodegenerative disorder caused by the accumulation of the GM₂ganglioside in the brain (Igdoura, S. A. et al. (1999) Hum. Mol. Genet.8:1111-6) due to a deficiency of the enzyme hexosaminidase A. Thedisease is characterized by the onset of developmental retardation,followed by paralysis, dementia, blindness, and usually death within thesecond or third year of life. Confirmatory evidence of Tay-Sachs diseaseis obtained at autopsy upon the identification of ballooned neurons inthe central nervous system (Online Mendelian Inheritance in Man (OMIM).Johns Hopkins University, Baltimore, Md. MIM Number: 272800, Aug. 4,2000, WWW URL: http://www.ncbi.nlm.nih.gov/omim/). In the case ofTay-Sachs disease, cortical pyramidal neurons undergo a second round ofdendritogenesis (Walkley, S. U. et al. (1998) Ann. N.Y. Acad. Sci.845:188-99).

[0056] Other diseases are also associated with defects in sialidaseactivity. G_(M1) gangliosidosis and Morquio B disease both arise frombeta-galactosidase deficiency, although the diseases present withdistinct phenotypes. Sialidosis arises from a neuraminidase deficiencybut presents with symptoms similar to gangliosidosis. A likely reasonfor the overlapping phenotypes of sialidase deficiencies is the presenceof these enzymes in a complex in lysosomes (Callahan, J. W. (1999)Biochim. Biophys. Acta 1455:85-103).

[0057] PLAs are implicated in a variety of disease processes. Forexample, PLAs are found in the pancreas, in cardiac tissue, and ininflammation-associated tissues. Pancreatic PLAs function in thedigestion of dietary lipids and have been proposed to play a role incell proliferation, smooth muscle contraction, and acute lung injury.Inflammatory PLAs are potent mediators of inflammatory processes and arehighly expressed in serum and synovial fluids of patients withinflammatory disorders. Additionally, inflammatory PLAs are found inmost human cell types and are expressed in diverse pathologicalprocesses such as septic shock, intestinal cancers, rheumatoidarthritis, and epidermal hyperplasia.

[0058] The role of PLBs in human tissues has been investigated invarious research studies. Hydrolysis of lysophosphatidylcholine by PLBscauses lysis in erythrocyte membranes (Selle, supra). Similarly,Endresen, M. J. et al. (1993; Scand. J. Clin. Invest. 53:733-739)reported that the increased hydrolysis of lysophosphatidylcholine by PLBin pre-eclamptic women causes release of free fatty acids into the sera.In renal studies, PLB was shown to protect Na⁺,K⁺-ATPase from thecytotoxic and cytolytic effects of cyclosporin A (Anderson, supra).

[0059] Lipases, phospholipases, and lipoxygenases are thought tocontribute to complex diseases, such as atherosclerosis, obesity,arthritis, asthma, and cancer, as well as to single gene defects, suchas Wolman's disease and Type I hyperlipoproteinemia.

[0060] The discovery of new lipid-associated molecules, and thepolynucleotides encoding them, satisfies a need in the art by providingnew compositions which are useful in the diagnosis, prevention, andtreatment of cancers, neurological, autoimmune/inflammatory,gastrointestinal, and cardiovascular disorders, and disorders of lipidmetabolism, and in the assessment of the effects of exogenous compoundson the expression of nucleic acid and amino acid sequences oflipid-associated molecules.

SUMMARY OF THE INVENTION

[0061] The invention features purified polypeptides, lipid-associatedmolecules, referred to collectively as “LIPAM” and individually as“LIPAM-1,” “LIPAM-2,” “LIPAM-3,” “LIPAM4,” “LIPAM-5,” “LIPAM-6,”“LIPAM-7,” “LIPAM-8,” and “LIPAM-9.” In one aspect, the inventionprovides an isolated polypeptide selected from the group consisting ofa) a polypeptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO:1-9, b) a polypeptide comprising anaturally occurring amino acid sequence at least 90% identical to anamino acid sequence selected from the group consisting of SEQ ID NO:1-9,c) a biologically active fragment of a polypeptide having an amino acidsequence selected from the group consisting of SEQ ID NO:1-9, and d) animmunogenic fragment of a polypeptide having an amino acid sequenceselected from the group consisting of SEQ ID NO:1-9. In one alternative,the invention provides an isolated polypeptide comprising the amino acidsequence of SEQ ID NO:1-9.

[0062] The invention further provides an isolated polynucleotideencoding a polypeptide selected from the group consisting of a) apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-9, b) a polypeptide comprising a naturallyoccurring amino acid sequence at least 90% identical to an amino acidsequence selected from the group consisting of SEQ ID NO:1-9, c) abiologically active fragment of a polypeptide having an amino acidsequence selected from the group consisting of SEQ ID NO:1-9, and d) animmunogenic fragment of a polypeptide having an amino acid sequenceselected from the group consisting of SEQ ID NO:1-9. In one alternative,the polynucleotide encodes a polypeptide selected from the groupconsisting of SEQ ID NO:1-9. In another alternative, the polynucleotideis selected from the group consisting of SEQ ID NO:10-18.

[0063] Additionally, the invention provides a recombinant polynucleotidecomprising a promoter sequence operably linked to a polynucleotideencoding a polypeptide selected from the group consisting of a) apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-9, b) a polypeptide comprising a naturallyoccurring amino acid sequence at least 90% identical to an amino acidsequence selected from the group consisting of SEQ ID NO:1-9, c) abiologically active fragment of a polypeptide having an amino acidsequence selected from the group consisting of SEQ ID NO:1-9, and d) animmunogenic fragment of a polypeptide having an amino acid sequenceselected from the group consisting of SEQ ID NO:1-9. In one alternative,the invention provides a cell transformed with the recombinantpolynucleotide. In another alternative, the invention provides atransgenic organism comprising the recombinant polynucleotide.

[0064] The invention also provides a method for producing a polypeptideselected from the group consisting of a) a polypeptide comprising anamino acid sequence selected from the group consisting of SEQ ID NO:1-9,b) a polypeptide comprising a naturally occurring amino acid sequence atleast 90% identical to an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-9, c) a biologically active fragment of apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-9, and d) an immunogenic fragment of apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-9. The method comprises a) culturing a cellunder conditions suitable for expression of the polypeptide, whereinsaid cell is transformed with a recombinant polynucleotide comprising apromoter sequence operably linked to a polynucleotide encoding thepolypeptide, and b) recovering the polypeptide so expressed.

[0065] Additionally, the invention provides an isolated antibody whichspecifically binds to a polypeptide selected from the group consistingof a) a polypeptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO:1-9, b) a polypeptide comprising anaturally occurring amino acid sequence at least 90% identical to anamino acid sequence selected from the group consisting of SEQ ID NO:1-9,c) a biologically active fragment of a polypeptide having an amino acidsequence selected from the group consisting of SEQ ID NO:1-9, and d) animmunogenic fragment of a polypeptide having an amino acid sequenceselected from the group consisting of SEQ ID NO:1-9.

[0066] The invention further provides an isolated polynucleotideselected from the group consisting of a) a polynucleotide comprising apolynucleotide sequence selected from the group consisting of SEQ IDNO:10-18, b) a polynucleotide comprising a naturally occurringpolynucleotide sequence at least 90% identical to a polynucleotidesequence selected from the group consisting of SEQ ID NO:10-18, c) apolynucleotide complementary to the polynucleotide of a), d) apolynucleotide complementary to the polynucleotide of b), and e) an RNAequivalent of a)-d). In one alternative, the polynucleotide comprises atleast 60 contiguous nucleotides.

[0067] Additionally, the invention provides a method for detecting atarget polynucleotide in a sample, said target polynucleotide having asequence of a polynucleotide selected from the group consisting of a) apolynucleotide comprising a polynucleotide sequence selected from thegroup consisting of SEQ ID NO:10-18, b) a polynucleotide comprising anaturally occurring polynucleotide sequence at least 90% identical to apolynucleotide sequence selected from the group consisting of SEQ IDNO:10-18, c) a polynucleotide complementary to the polynucleotide of a),d) a polynucleotide complementary to the polynucleotide of b), and e) anRNA equivalent of a)-d). The method comprises a) hybridizing the samplewith a probe comprising at least 20 contiguous nucleotides comprising asequence complementary to said target polynucleotide in the sample, andwhich probe specifically hybridizes to said target polynucleotide, underconditions whereby a hybridization complex is formed between said probeand said target polynucleotide or fragments thereof, and b) detectingthe presence or absence of said hybridization complex, and optionally,if present, the amount thereof. In one alternative, the probe comprisesat least 60 contiguous nucleotides.

[0068] The invention further provides a method for detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide selected from the group consisting of a) apolynucleotide comprising a polynucleotide sequence selected from thegroup consisting of SEQ ID NO:10-18, b) a polynucleotide comprising anaturally occurring polynucleotide sequence at least 90% identical to apolynucleotide sequence selected from the group consisting of SEQ IDNO:10-18, c) a polynucleotide complementary to the polynucleotide of a),d) a polynucleotide complementary to the polynucleotide of b), and e) anRNA equivalent of a)-d). The method comprises a) amplifying said targetpolynucleotide or fragment thereof using polymerase chain reactionamplification, and b) detecting the presence or absence of saidamplified target polynucleotide or fragment thereof, and, optionally, ifpresent, the amount thereof.

[0069] The invention further provides a composition comprising aneffective amount of a polypeptide selected from the group consisting ofa) a polypeptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO:1-9, b) a polypeptide comprising anaturally occurring amino acid sequence at least 90% identical to anamino acid sequence selected from the group consisting of SEQ ID NO:1-9,c) a biologically active fragment of a polypeptide having an amino acidsequence selected from the group consisting of SEQ ID NO:1-9, and d) animmunogenic fragment of a polypeptide having an amino acid sequenceselected from the group consisting of SEQ ID NO: 1-9, and apharmaceutically acceptable excipient. In one embodiment, thecomposition comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-9. The invention additionally provides amethod of treating a disease or condition associated with decreasedexpression of functional LIPAM, comprising administering to a patient inneed of such treatment the composition.

[0070] The invention also provides a method for screening a compound foreffectiveness as an agonist of a polypeptide selected from the groupconsisting of a) a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 1-9, b) a polypeptidecomprising a naturally occurring amino acid sequence at least 90%identical to an amino acid sequence selected from the group consistingof SEQ ID NO:1-9, c) a biologically active fragment of a polypeptidehaving an amino acid sequence selected from the group consisting of SEQID NO:1-9, and d) an immunogenic fragment of a polypeptide having anamino acid sequence selected from the group consisting of SEQ ID NO:1-9.The method comprises a) exposing a sample comprising the polypeptide toa compound, and b) detecting agonist activity in the sample. In onealternative, the invention provides a composition comprising an agonistcompound identified by the method and a pharmaceutically acceptableexcipient. In another alternative, the invention provides a method oftreating a disease or condition associated with decreased expression offunctional LIPAM, comprising administering to a patient in need of suchtreatment the composition.

[0071] Additionally, the invention provides a method for screening acompound for effectiveness as an antagonist of a polypeptide selectedfrom the group consisting of a) a polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NO:1-9, b) apolypeptide comprising a naturally occurring amino acid sequence atleast 90% identical to an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-9, c) a biologically active fragment of apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-9, and d) an immunogenic fragment of apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-9. The method comprises a) exposing a samplecomprising the polypeptide to a compound, and b) detecting antagonistactivity in the sample. In one alternative, the invention provides acomposition comprising an antagonist compound identified by the methodand a pharmaceutically acceptable excipient. In another alternative, theinvention provides a method of treating a disease or conditionassociated with overexpression of functional LIPAM, comprisingadministering to a patient in need of such treatment the composition.

[0072] The invention further provides a method of screening for acompound that specifically binds to a polypeptide selected from thegroup consisting of a) a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1-9, b) a polypeptidecomprising a naturally occurring amino acid sequence at least 90%identical to an amino acid sequence selected from the group consistingof SEQ ID NO:1-9, c) a biologically active fragment of a polypeptidehaving an amino acid sequence selected from the group consisting of SEQID NO:1-9, and d) an immunogenic fragment of a polypeptide having anamino acid sequence selected from the group consisting of SEQ ID NO:1-9.The method comprises a) combining the polypeptide with at least one testcompound under suitable conditions, and b) detecting binding of thepolypeptide to the test compound, thereby identifying a compound thatspecifically binds to the polypeptide.

[0073] The invention further provides a method of screening for acompound that modulates the activity of a polypeptide selected from thegroup consisting of a) a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1-9, b) a polypeptidecomprising a naturally occurring amino acid sequence at least 90%identical to an amino acid sequence selected from the group consistingof SEQ ID NO:1-9, c) a biologically active fragment of a polypeptidehaving an amino acid sequence selected from the group consisting of SEQID NO:1-9, and d) an immunogenic fragment of a polypeptide having anamino acid sequence selected from the group consisting of SEQ ID NO:1-9.The method comprises a) combining the polypeptide with at least one testcompound under conditions permissive for the activity of thepolypeptide, b) assessing the activity of the polypeptide in thepresence of the test compound, and c) comparing the activity of thepolypeptide in the presence of the test compound with the activity ofthe polypeptide in th absence of the test compound, wherein a change inthe activity of the polypeptide in the presence of the test compound isindicative of a compound that modulates the activity of the polypeptide.

[0074] The invention further provides a method for screening a compoundfor effectiveness in altering expression of a target polynucleotide,wherein said target polynucleotide comprises a polynucleotide sequenceselected from the group consisting of SEQ ID NO:10-18, the methodcomprising a) exposing a sample comprising the target polynucleotide toa compound, b) detecting altered expression of the targetpolynucleotide, and c) comparing the expression of the targetpolynucleotide in the presence of varying amounts of the compound and inthe absence of the compound.

[0075] The invention further provides a method for assessing toxicity ofa test compound, said method comprising a) treating a biological samplecontaining nucleic acids with the test compound; b) hybridizing thenucleic acids of the treated biological sample with a probe comprisingat least 20 contiguous nucleotides of a polynucleotide selected from thegroup consisting of i) a polynucleotide comprising a polynucleotidesequence selected from the group consisting of SEQ ID NO:10-18, ii) apolynucleotide comprising a naturally occurring polynucleotide sequenceat least 90% identical to a polynucleotide sequence selected from thegroup consisting of SEQ ID NO:10-18, iii) a polynucleotide having asequence complementary to i), iv) a polynucleotide complementary to thepolynucleotide of ii), and v) an RNA equivalent of i)-iv). Hybridizationoccurs under conditions whereby a specific hybridization complex isformed between said probe and a target polynucleotide in the biologicalsample, said target polynucleotide selected from the group consisting ofi) a polynucleotide comprising a polynucleotide sequence selected fromthe group consisting of SEQ ID NO:10-18, ii) a polynucleotide comprisinga naturally occurring polynucleotide sequence at least 90% identical toa polynucleotide sequence selected from the group consisting of SEQ IDNO:10-18, iii) a polynucleotide complementary to the polynucleotide ofi), iv) a polynucleotide complementary to the polynucleotide of ii), andv) an RNA equivalent of i)-iv). Alternatively, the target polynucleotidecomprises a fragment of a polynucleotide sequence selected from thegroup consisting of i)-v) above; c) quantifying the amount ofhybridization complex; and d) comparing the amount of hybridizationcomplex in the treated biological sample with the amount ofhybridization complex in an untreated biological sample, wherein adifference in the amount of hybridization complex in the treatedbiological sample is indicative of toxicity of the test compound.

BRIEF DESCRIPTION OF THE TABLES

[0076] Table 1 summarizes the nomenclature for the full lengthpolynucleotide and polypeptide sequences of the present invention.

[0077] Table 2 shows the GenBank identification number and annotation ofthe nearest GenBank homolog for polypeptides of the invention. Theprobability scores for the matches between each polypeptide and itshomolog(s) are also shown.

[0078] Table 3 shows structural features of polypeptide sequences of theinvention, including predicted motifs and domains, along with themethods, algorithms, and searchable databases used for analysis of thepolypeptides.

[0079] Table 4 lists the cDNA and/or genomic DNA fragments which wereused to assemble polynucleotide sequences of the invention, along withselected fragments of the polynucleotide sequences.

[0080] Table 5 shows the representative cDNA library for polynucleotidesof the invention.

[0081] Table 6 provides an appendix which describes the tissues andvectors used for construction of the cDNA libraries shown in Table 5.

[0082] Table 7 shows the tools, programs, and algorithms used to analyzethe polynucleotides and polypeptides of the invention, along withapplicable descriptions, references, and threshold parameters.

DESCRIPTION OF THE INVENTION

[0083] Before the present proteins, nucleotide sequences, and methodsare described, it is understood that this invention is not limited tothe particular machines, materials and methods described, as these mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention which will belimited only by the appended claims.

[0084] It must be noted that as used herein and in the appended claims,the singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, a referenceto “a host cell” includes a plurality of such host cells, and areference to “an antibody” is a reference to one or more antibodies andequivalents thereof known to those skilled in the art, and so forth.

[0085] Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any machines,materials, and methods similar or equivalent to those described hereincan be used to practice or test the present invention, the preferredmachines, materials and methods are now described. All publicationsmentioned herein are cited for the purpose of describing and disclosingthe cell lines, protocols, reagents and vectors which are reported inthe publications and which might be used in connection with theinvention. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

[0086] Definitions

[0087] “LIPAM” refers to the amino acid sequences of substantiallypurified LIPAM obtained from any species, particularly a mammalianspecies, including bovine, ovine, porcine, murine, equine, and human,and from any source, whether natural, synthetic, semi-synthetic, orrecombinant.

[0088] The term “agonist” refers to a molecule which intensifies ormimics the biological activity of LIPAM. Agonists may include proteins,nucleic acids, carbohydrates, small molecules, or any other compound orcomposition which modulates the activity of LIPAM either by directlyinteracting with LIPAM or by acting on components of the biologicalpathway in which LIPAM participates.

[0089] An “allelic variant” is an alternative form of the gene encodingLIPAM. Allelic variants may result from at least one mutation in thenucleic acid sequence and may result in altered mRNAs or in polypeptideswhose structure or function may or may not be altered. A gene may havenone, one, or many allelic variants of its naturally occurring form.Common mutational changes which give rise to allelic variants aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

[0090] “Altered” nucleic acid sequences encoding LIPAM include thosesequences with deletions, insertions, or substitutions of differentnucleotides, resulting in a polypeptide the same as LIPAM or apolypeptide with at least one-functional characteristic of LIPAM.Included within this definition are polymorphisms which may or may notbe readily detectable using a particular oligonucleotide probe of thepolynucleotide encoding LIPAM, and improper or unexpected hybridizationto allelic variants, with a locus other than the normal chromosomallocus for the polynucleotide sequence encoding LIPAM. The encodedprotein may also be “altered,” and may contain deletions, insertions, orsubstitutions of amino acid residues which produce a silent change andresult in a functionally equivalent LIPAM. Deliberate amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues, as long as the biological orimmunological activity of LIPAM is retained. For example, negativelycharged amino acids may include aspartic acid and glutamic acid, andpositively charged amino acids may include lysine and arginine. Aminoacids with uncharged polar side chains having similar hydrophilicityvalues may include: asparagine and glutamine; and serine and threonine.Amino acids with uncharged side chains having similar hydrophilicityvalues may include: leucine, isoleucine, and valine; glycine andalanine; and phenylalanine and tyrosine.

[0091] The terms “amino acid” and “amino acid sequence” refer to anoligopeptide, peptide, polypeptide, or protein sequence, or a fragmentof any of these, and to naturally occurring or synthetic molecules.Where “amino acid sequence” is recited to refer to a sequence of anaturally occurring protein molecule, “amino acid sequence” and liketerms are not meant to limit the amino acid sequence to the completenative amino acid sequence associated with the recited protein molecule.

[0092] “Amplification” relates to the production of additional copies ofa nucleic acid sequence. Amplification is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art.

[0093] The term “antagonist” refers to a molecule which inhibits orattenuates the biological activity of LIPAM. Antagonists may includeproteins such as antibodies, nucleic acids, carbohydrates, smallmolecules, or any other compound or composition which modulates theactivity of LIPAM either by directly interacting with LIPAM or by actingon components of the biological pathway in which LIPAM participates.

[0094] The term “antibody” refers to intact immunoglobulin molecules aswell as to fragments thereof, such as Fab, F(ab′)₂, and Fv fragments,which are capable of binding an epitopic determinant. Antibodies thatbind LIPAM polypeptides can be prepared using intact polypeptides orusing fragments containing small peptides of interest as the immunizingantigen. The polypeptide or oligopeptide used to immunize an animal(e.g., a mouse, a rat, or a rabbit) can be derived from the translationof RNA, or synthesized chemically, and can be conjugated to a carrierprotein if desired. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin, thyroglobulin, and keyholelimpet hemocyanin (KLH). The coupled peptide is then used to immunizethe animal.

[0095] The term “antigenic determinant” refers to that region of amolecule (i.e., an epitope) that makes contact with a particularantibody. When a protein or a fragment of a protein is used to immunizea host animal, numerous regions of the protein may induce the productionof antibodies which bind specifically to antigenic determinants(particular regions or three-dimensional structures on the protein). Anantigenic determinant may compete with the intact antigen (i.e., theimmunogen used to elicit the immune response) for binding to anantibody.

[0096] The term “aptamer” refers to a nucleic acid or oligonucleotidemolecule that binds to a specific molecular target. Aptamers are derivedfrom an in vitro evolutionary process (e.g., SELEX (Systematic Evolutionof Ligands by EXponential Enrichment), described in U.S. Pat. No.5,270,163), which selects for target-specific aptamer sequences fromlarge combinatorial libraries. Aptamer compositions may bedouble-stranded or single-stranded, and may includedeoxyribonucleotides, ribonucleotides, nucleotide derivatives, or othernucleotide-like molecules. The nucleotide components of an aptamer mayhave modified sugar groups (e.g., the 2′-OH group of a ribonucleotidemay be replaced by 2′-F or 2′-NH₂), which may improve a desiredproperty, e.g., resistance to nucleases or longer lifetime in blood.Aptamers may be conjugated to other molecules, e.g., a high molecularweight carrier to slow clearance of the aptamer from the circulatorysystem. Aptamers may be specifically cross-linked to their cognateligands, e.g., by photo-activation of a cross-linker. (See, e.g., Brody,E. N. and L. Gold (2000) J. Biotechnol. 74:5-13.)

[0097] The term “intramer” refers to an aptamer which is expressed invivo. For example, a vaccinia virus-based RNA expression system has beenused to express specific RNA aptamers at high levels in the cytoplasm ofleukocytes (Blind, M. et al. (1999) Proc. Natl Acad. Sci. USA96:3606-3610).

[0098] The term “spiegelmer” refers to an aptamer which includes L-DNA,L-RNA, or other left-handed nucleotide derivatives or nucleotide-likemolecules. Aptamers containing left-handed nucleotides are resistant todegradation by naturally occurring enzymes, which normally act onsubstrates containing right-handed nucleotides.

[0099] The term “antisense” refers to any composition capable ofbase-pairing with the “sense” (coding) strand of a specific nucleic acidsequence. Antisense compositions may include DNA; RNA; peptide nucleicacid (PNA); oligonucleotides having modified backbone linkages such asphosphorothioates, methylphosphonates, or benzylphosphonates;oligonucleotides having modified sugar groups such as 2′-methoxyethylsugars or 2′-methoxyethoxy sugars; or oligonucleotides having modifiedbases such as 5-methyl cytosine, 2′-deoxyuracil, or7-deaza-2′-deoxyguanosine. Antisense molecules may be produced by anymethod including chemical synthesis or transcription. Once introducedinto a cell, the complementary antisense molecule base-pairs with anaturally occurring nucleic acid sequence produced by the cell to formduplexes which block either transcription or translation. Thedesignation “negative” or “minus” can refer to the antisense strand, andthe designation “positive” or “plus” can refer to the sense strand of areference DNA molecule.

[0100] The term “biologically active” refers to a protein havingstructural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, “immunologically active” or “immunogenic”refers to the capability of the natural, recombinant, or syntheticLIPAM, or of any oligopeptide thereof, to induce a specific immuneresponse in appropriate animals or cells and to bind with specificantibodies.

[0101] “Complementary” describes the relationship between twosingle-stranded nucleic acid sequences that anneal by base-pairing. Forexample, 5′-AGT-3′ pairs with its complement, 3′-TCA-5′.

[0102] A “composition comprising a given polynucleotide sequence” and a“composition comprising a given amino acid sequence” refer broadly toany composition containing the given polynucleotide or amino acidsequence. The composition may comprise a dry formulation or an aqueoussolution. Compositions comprising polynucleotide sequences encodingLIPAM or fragments of LIPAM may be employed as hybridization probes. Theprobes may be stored in freeze-dried form and may be associated with astabilizing agent such as a carbohydrate. In hybridizations, the probemay be deployed in an aqueous solution containing salts (e.g., NaCl),detergents (e.g., sodium dodecyl sulfate; SDS), and other components(e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).

[0103] “Consensus sequence” refers to a nucleic acid sequence which hasbeen subjected to repeated DNA sequence analysis to resolve uncalledbases, extended using the XL,PCR kit (Applied Biosystems, Foster CityCalif.) in the 5′ and/or the 3′ direction, and resequenced, or which hasbeen assembled from one or more overlapping cDNA, EST, or genomic DNAfragments using a computer program for fragment assembly, such as theGELVIEW fragment assembly system (GCG, Madison Wis.) or Phrap(University of Washington, Seattle Wash.). Some sequences have been bothextended and assembled to produce the consensus sequence.

[0104] “Conservative amino acid substitutions” are those substitutionsthat are predicted to least interfere with the properties of theoriginal protein, i.e., the structure and especially the function of theprotein is conserved and not significantly changed by suchsubstitutions. The table below shows amino acids which may besubstituted for an original amino acid in a protein and which areregarded as conservative amino acid substitutions. Original ResidueConservative Substitution Ala Gly, Ser Arg His, Lys Asn Asp, Gin, HisAsp Asn, Glu Cys Ala, Ser Gln Asn, Glu, His Glu Asp, Gln, His Gly AlaHis Asn, Arg, Gln, Glu Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu MetLeu, Ile Phe His, Met, Leu, Trp, Tyr Ser Cys, Thr Thr Ser, Val Trp Phe,Tyr Tyr His, Phe, Trp Val Ile, Leu, Thr

[0105] Conservative amino acid substitutions generally maintain (a) thestructure of the polypeptide backbone in the area of the substitution,for example, as a beta sheet or alpha helical conformation, (b) thecharge or hydrophobicity of the molecule at the site of thesubstitution, and/or (c) the bulk of the side chain.

[0106] A “deletion” refers to a change in the amino acid or nucleotidesequence that results in the absence of one or more amino acid residuesor nucleotides.

[0107] The term “derivative” refers to a chemically modifiedpolynucleotide or polypeptide. Chemical modifications of apolynucleotide can include, for example, replacement of hydrogen by analkyl, acyl, hydroxyl, or amino group. A derivative polynucleotideencodes a polypeptide which retains at least one biological orimmunological function of the natural molecule. A derivative polypeptideis one modified by glycosylation, pegylation, or any similar processthat retains at least one biological or immunological function of thepolypeptide from which it was derived.

[0108] A “detectable label” refers to a reporter molecule or enzyme thatis capable of generating a measurable signal and is covalently ornoncovalently joined to a polynucleotide or polypeptide.

[0109] “Differential expression” refers to increased or upregulated; ordecreased, downregulated, or absent gene or protein expression,determined by comparing at least two different samples. Such comparisonsmay be carried out between, for example, a treated and an untreatedsample, or a diseased and a normal sample.

[0110] “Exon shuffling” refers to the recombination of different codingregions (exons). Since an exon may represent a structural or functionaldomain of the encoded protein, new proteins may be assembled through thenovel reassortment of stable substructures, thus allowing accelerationof the evolution of new protein functions.

[0111] A “fragment” is a unique portion of LIPAM or the polynucleotideencoding LIPAM which is identical in sequence to but shorter in lengththan the parent sequence. A fragment may comprise up to the entirelength of the defined sequence, minus one nucleotide/amino acid residue.For example, a fragment may comprise from 5 to 1000 contiguousnucleotides or amino acid residues. A fragment used as a probe, primer,antigen, therapeutic molecule, or for other purposes, may be at least 5,10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500contiguous nucleotides or amino acid residues in length. Fragments maybe preferentially selected from certain regions of a molecule. Forexample, a polypeptide fragment may comprise a certain length ofcontiguous amino acids selected from the first 250 or 500 amino acids(or first 25% or 50%) of a polypeptide as shown in a certain definedsequence. Clearly these lengths are exemplary, and any length that issupported by the *specification, including the Sequence Listing, tables,and figures, may be encompassed by the present embodiments.

[0112] A fragment of SEQ ID NO:10-18 comprises a region of uniquepolynucleotide sequence that specifically identifies SEQ ID NO:10-18,for example, as distinct from any other sequence in the genome fromwhich the fragment was obtained. A fragment of SEQ ID NO:10-18 isuseful, for example, in hybridization and amplification technologies andin analogous methods that distinguish SEQ ID NO:10-18 from relatedpolynucleotide sequences. The precise length of a fragment of SEQ IDNO:10-18 and the region of SEQ ID NO:10-18 to which the fragmentcorresponds are routinely determinable by one of ordinary skill in theart based on the intended purpose for the fragment.

[0113] A fragment of SEQ ID NO:1-9 is encoded by a fragment of SEQ IDNO:10-18. A fragment of SEQ ID NO:1-9 comprises a region of unique aminoacid sequence that specifically identifies SEQ ID NO:1-9. For example, afragment of SEQ ID NO:1-9 is useful as an immunogenic peptide for thedevelopment of antibodies that specifically recognize SEQ ID NO:1-9. Theprecise length of a fragment of SEQ ID NO:1-9 and the region of SEQ IDNO:1-9 to which the fragment corresponds are routinely determinable byone of ordinary skill in the art based on the intended purpose for thefragment.

[0114] A “full length” polynucleotide sequence is one containing atleast a translation initiation codon (e.g., methionine) followed by anopen reading frame and a translation termination codon. A “full length”polynucleotide sequence encodes a “full length” polypeptide sequence.

[0115] “Homology” refers to sequence similarity or, interchangeably,sequence identity, between two or more polynucleotide sequences or twoor more polypeptide sequences.

[0116] The terms “percent identity” and “% identity,” as applied topolynucleotide sequences, refer to the percentage of residue matchesbetween at least two polynucleotide sequences aligned using astandardized algorithm. Such an algorithm may insert, in a standardizedand reproducible way, gaps in the sequences being compared in order tooptimize alignment between two sequences, and therefore achieve a moremeaningful comparison of the two sequences.

[0117] Percent identity between polynucleotide sequences may bedetermined using the default parameters of the CLUSTAL V algorithm asincorporated into the MEGALIGN version 3.12e sequence alignment program.This program is part of the LASERGENE software package, a suite ofmolecular biological analysis programs (DNASTAR, Madison Wis.). CLUSTALV is described in Higgins, D. G. and P. M. Sharp (1989) CABIOS 5:151-153and in Higgins, D. G. et al. (1992) CABIOS 8:189-191. For pairwisealignments of polynucleotide sequences, the default parameters are setas follows: Ktuple=2, gap penalty=5, window=4, and “diagonals saved”=4.The “weighted” residue weight table is selected as the default. Percentidentity is reported by CLUSTAL V as the “percent similarity” betweenaligned polynucleotide sequences.

[0118] Alternatively, a suite of commonly used and freely availablesequence comparison algorithms is provided by the National Center forBiotechnology Information (NCBI) Basic Local Alignment Search Tool(BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403-410), whichis available from several sources, including the NCBI, Bethesda, Md.,and on the Internet at http://www.ncbi.nlm.nih.gov/BLAST/. The BLASTsoftware suite includes various sequence analysis programs including“blastn,” that is used to align a known polynucleotide sequence withother polynucleotide sequences from a variety of databases. Alsoavailable is a tool called “BLAST 2 Sequences” that is used for directpairwise comparison of two nucleotide sequences. “BLAST 2 Sequences” canbe accessed and used interactively athttp://www.ncbi.nlm.nih.gov/gorf/bl2.html. The “BLAST 2 Sequences” toolcan be used for both blastn and blastp (discussed below). BLAST programsare commonly used with gap and other parameters set to default settings.For example, to compare two nucleotide sequences, one may use blastnwith the “BLAST 2 Sequences” tool Version 2.0.12 (Apr. 21, 2000) set atdefault parameters. Such default parameters may be, for example:

[0119] Matrix: BLOSUM62

[0120] Reward for match: 1

[0121] Penalty for mismatch: −2

[0122] Open Gap: 5 and Extension Gap: 2 penalties

[0123] Gap×drop-off: 50

[0124] Expect: 10

[0125] Word Size: 11

[0126] Filter: on

[0127] Percent identity may be measured over the length of an entiredefined sequence, for example, as defined by a particular SEQ ID number,or may be measured over a shorter length, for example, over the lengthof a fragment taken from a larger, defined sequence, for instance, afragment of at least 20, at least 30, at least 40, at least 50, at least70, at least 100, or at least 200 contiguous nucleotides. Such lengthsare exemplary only, and it is understood that any fragment lengthsupported by the sequences shown herein, in the tables, figures, orSequence Listing, may be used to describe a length over which percentageidentity may be measured.

[0128] Nucleic acid sequences that do not show a high degree of identitymay nevertheless encode similar amino acid sequences due to thedegeneracy of the genetic code. It is understood that changes in anucleic acid sequence can be made using this degeneracy to producemultiple nucleic acid sequences that all encode substantially the sameprotein.

[0129] The phrases “percent identity” and “% identity,” as applied topolypeptide sequences, refer to the percentage of residue matchesbetween at least two polypeptide sequences aligned using a standardizedalgorithm. Methods of polypeptide sequence alignment are well-known.Some alignment methods take into account conservative amino acidsubstitutions. Such conservative substitutions, explained in more detailabove, generally preserve the charge and hydrophobicity at the site ofsubstitution, thus preserving the structure (and therefore function) ofthe polypeptide.

[0130] Percent identity between polypeptide sequences may be determinedusing the default parameters of the CLUSTAL V algorithm as incorporatedinto the MEGALIGN version 3.12e sequence alignment program (describedand referenced above). For pairwise alignments of polypeptide sequencesusing CLUSTAL V, the default parameters are set as follows: Ktuple=1,gap penalty=3, window=5, and “diagonals saved”=5. The PAM250 matrix isselected as the default residue weight table. As with polynucleotidealignments, the percent identity is reported by CLUSTAL V as the“percent similarity” between aligned polypeptide sequence pairs.

[0131] Alternatively the NCBI BLAST software suite may be used. Forexample, for a pairwise comparison of two polypeptide sequences, one mayuse the “BLAST 2 Sequences” tool Version 2.0.12 (Apr. 21, 2000) withblastp set at default parameters. Such default parameters may be, forexample:

[0132] Matrix: BLOSUM62

[0133] Open Gap: 11 and Extension Gap: 1 penalties

[0134] Gap×drop-off: 50

[0135] Expect: 10

[0136] Word Size: 3

[0137] Filter: on

[0138] Percent identity may be measured over the length of an entiredefined polypeptide sequence, for example, as defined by a particularSEQ ID number, or may be measured over a shorter length, for example,over the length of a fragment taken from a larger, defined polypeptidesequence, for instance, a fragment of at least 15, at least 20, at least30, at least 40, at least 50, at least 70 or at least 150 contiguousresidues. Such lengths are exemplary only, and it is understood that anyfragment length supported by the sequences shown herein, in the tables,figures or Sequence Listing, may be used to describe a length over whichpercentage identity may be measured.

[0139] “Human artificial chromosomes” (HACs) are linear microchromosomeswhich may contain DNA sequences of about 6 kb to 10 Mb in size and whichcontain all of the elements required for chromosome replication,segregation and maintenance.

[0140] The term “humanized antibody” refers to an antibody molecule inwhich the amino acid sequence in the non-antigen binding regions hasbeen altered so that the antibody more closely resembles a humanantibody, and still retains its original binding ability.

[0141] “Hybridization” refers to the process by which a polynucleotidestrand anneals with a complementary strand through base pairing underdefined hybridization conditions. Specific hybridization is anindication that two nucleic acid sequences share a high degree ofcomplementarity. Specific hybridization complexes form under permissiveannealing conditions and remain hybridized after the “washing” step(s).The washing step(s) is particularly important in determining thestringency of the hybridization process, with more stringent conditionsallowing less non-specific binding, i.e., binding between pairs ofnucleic acid strands that are not perfectly matched. Permissiveconditions for annealing of nucleic acid sequences are routinelydeterminable by one of ordinary skill in the art and may be consistentamong hybridization experiments, whereas wash conditions may be variedamong experiments to achieve the desired stringency, and thereforehybridization specificity. Permissive annealing conditions occur, forexample, at 68° C. in the presence of about 6×SSC, about 1% (w/v) SDS,and about 100 μg/ml sheared, denatured salmon sperm DNA.

[0142] Generally, stringency of hybridization is expressed, in part,with reference to the temperature under which the wash step is carriedout. Such wash temperatures are typically selected to be about 5° C. to20° C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. An equation forcalculating T_(m) and conditions for nucleic acid hybridization are wellknown and can be found in Sambrook, J. et al. (1989) Molecular Cloning:A Laboratory Manual, 2^(n) ed., vol. 1-3, Cold Spring Harbor Press,Plainview N.Y.; specifically see volume 2, chapter 9.

[0143] High stringency conditions for hybridization betweenpolynucleotides of the present invention include wash conditions of 68°C. in the presence of about 0.2×SSC and about 0.1% SDS, for 1 hour.Alternatively, temperatures of about 65° C., 60° C., 55° C., or 42° C.may be used. SSC concentration may be varied from about 0.1 to 2×SSC,with SDS being present at about 0.1%. Typically, blocking reagents areused to block non-specific hybridization. Such blocking reagentsinclude, for instance, sheared and denatured salmon sperm DNA at about100-200 μg/ml. Organic solvent, such as formamide at a concentration ofabout 35-50% v/v, may also be used under particular circumstances, suchas for RNA:DNA hybridizations. Useful variations on these washconditions will be readily apparent to those of ordinary skill in theart. Hybridization, particularly under high stringency conditions, maybe suggestive of evolutionary similarity between the nucleotides. Suchsimilarity is strongly indicative of a similar role for the nucleotidesand their encoded polypeptides.

[0144] The term “hybridization complex” refers to a complex formedbetween two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary bases. A hybridization complex maybe formed in solution (e.g., C₀t or R₀t analysis) or formed between onenucleic acid sequence present in solution and another nucleic acidsequence immobilized on a solid support (e.g., paper, membranes,filters, chips, pins or glass slides, or any other appropriate substrateto which cells or their nucleic acids have been fixed).

[0145] The words “insertion” and “addition” refer to changes in an aminoacid or nucleotide sequence resulting in the addition of one or moreamino acid residues or nucleotides, respectively.

[0146] “Immune response” can refer to conditions associated withinflammation, trauma, immune disorders, or infectious or geneticdisease, etc. These conditions can be characterized by expression ofvarious factors, e.g., cytokines, chemokines, and other signalingmolecules, which may affect cellular and systemic defense systems.

[0147] An “immunogenic fragment” is a polypeptide or oligopeptidefragment of LIPAM which is capable of eliciting an immune response whenintroduced into a living organism, for example, a mammal. The term“immunogenic fragment” also includes any polypeptide or oligopeptidefragment of LIPAM which is useful in any of the antibody productionmethods disclosed herein or known in the art.

[0148] The term “microarray” refers to an arrangement of a plurality ofpolynucleotides, polypeptides, or other chemical compounds on asubstrate.

[0149] The terms “element” and “array element” refer to apolynucleotide, polypeptide, or other chemical compound having a uniqueand defined position on a microarray.

[0150] The term “modulate” refers to a change in the activity of LIPAM.For example, modulation may cause an increase or a decrease in proteinactivity, binding characteristics, or any other biological, functional,or immunological properties of LIPAM.

[0151] The phrases “nucleic acid” and “nucleic acid sequence” refer to anucleotide, oligonucleotide, polynucleotide, or any fragment thereof.These phrases also refer to DNA or RNA of genomic or syntheticorigin-which may be single-stranded or double-stranded and may representthe sense or the antisense strand, to peptide nucleic acid (PNA), or toany DNA-like or RNA-like material.

[0152] “Operably linked” refers to the situation in which a firstnucleic acid sequence is placed in a functional relationship with asecond nucleic acid sequence. For instance, a promoter is operablylinked to a coding sequence if the promoter affects the transcription orexpression of the coding sequence. Operably linked DNA sequences may bein close proximity or contiguous and, where necessary to join twoprotein coding regions, in the same reading frame.

[0153] “Peptide nucleic acid” (PNA) refers to an antisense molecule oranti-gene agent which comprises an oligonucleotide of at least about 5nucleotides in length linked to a peptide backbone of amino acidresidues ending in lysine. The terminal lysine confers solubility to thecomposition. PNAs preferentially bind complementary single stranded DNAor RNA and stop transcript elongation, and may be pegylated to extendtheir lifespan in the cell.

[0154] “Post-translational modification” of an LIPAM may involvelipidation, glycosylation, phosphorylation, acetylation, racemization,proteolytic cleavage, and other modifications known in the art. Theseprocesses may occur synthetically or biochemically. Biochemicalmodifications will vary by cell type depending on the enzymatic milieuof LIPAM.

[0155] “Probe” refers to nucleic acid sequences encoding LIPAM, theircomplements, or fragments thereof, which are used to detect identical,allelic or related nucleic acid sequences. Probes are isolatedoligonucleotides or polynucleotides attached to a detectable label orreporter molecule. Typical labels include radioactive isotopes, ligands,chemiluminescent agents, and enzymes. “Primers” are short nucleic acids,usually DNA oligonucleotides, which may be annealed to a targetpolynucleotide by complementary base-pairing. The primer may then beextended along the target DNA strand by a DNA polymerase enzyme. Primerpairs can be used for amplification (and identification) of a nucleicacid sequence, e.g., by the polymerase chain reaction (PCR).

[0156] Probes and primers as used in the present invention typicallycomprise at least 15 contiguous nucleotides of a known sequence. Inorder to enhance specificity, longer probes and primers may also beemployed, such as probes and primers that comprise at least 20, 25, 30,40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides ofthe disclosed nucleic acid sequences. Probes and primers may beconsiderably longer than these examples, and it is understood that anylength supported by the specification, including the tables, figures,and Sequence Listing, may be used.

[0157] Methods for preparing and using probes and primers are describedin the references, for example Sambrook, J. et al. (1989) MolecularCloning: A Laboratory Manual, 2^(n) ed., vol. 1-3, Cold Spring HarborPress, Plainview N.Y.; Ausubel, F. M. et al. (1987) Current Protocols inMolecular Biology, Greene Publ. Assoc. & Wiley-Intersciences, New YorkN.Y.; Innis, M. et al. (1990) PCR Protocols, A Guide to Methods andApplications, Academic Press, San Diego Calif. PCR primer pairs can bederived from a known sequence, for example, by using computer programsintended for that purpose such as Primer (Version 0.5, 1991, WhiteheadInstitute for Biomedical Research, Cambridge Mass.).

[0158] Oligonucleotides for use as primers are selected using softwareknown in the art for such purpose. For example, OLIGO 4.06 software isuseful for the selection of PCR primer pairs of up to 100 nucleotideseach, and for the analysis of oligonucleotides and largerpolynucleotides of up to 5,000 nucleotides from an input polynucleotidesequence of up to 32 kilobases. Similar primer selection programs haveincorporated additional features for expanded capabilities. For example,the PrimOU primer selection program (available to the public from theGenome Center at University of Texas South West Medical Center, DallasTex.) is capable of choosing specific primers from megabase sequencesand is thus useful for designing primers on a genome-wide scope. ThePrimer3 primer selection program (available to the public from theWhitehead Institute/MIT Center for Genome Research, Cambridge Mass.)allows the user to input a “mispriming library,” in which sequences toavoid as primer binding sites are user-specified. Primer3 is useful, inparticular, for the selection of oligonucleotides for microarrays. (Thesource code for the latter two primer selection programs may also beobtained from their respective sources and modified to meet the user'sspecific needs.) The PrimeGen program (available to the public from theUK Human Genome Mapping Project Resource Centre, Cambridge UK) designsprimers based on multiple sequence alignments, thereby allowingselection of primers that hybridize to either the most conserved orleast conserved regions of aligned nucleic acid sequences. Hence, thisprogram is useful for identification of both unique and conservedoligonucleotides and polynucleotide fragments. The oligonucleotides andpolynucleotide fragments identified by any of the above selectionmethods are useful in hybridization technologies, for example, as PCR orsequencing primers, microarray elements, or specific probes to identifyfully or partially complementary polynucleotides in a sample of nucleicacids. Methods of oligonucleotide selection are not limited to thosedescribed above.

[0159] A “recombinant nucleic acid” is a sequence that is not naturallyoccurring or has a sequence that is made by an artificial combination oftwo or more otherwise separated segments of sequence. This artificialcombination is often accomplished by chemical synthesis or, morecommonly, by the artificial manipulation of isolated segments of nucleicacids, e.g., by genetic engineering techniques such as those describedin Sambrook, supra. The term recombinant includes nucleic acids thathave been altered solely by addition, substitution, or deletion of aportion of the nucleic acid. Frequently, a recombinant nucleic acid mayinclude a nucleic acid sequence operably linked to a promoter sequence.Such a recombinant nucleic acid may be part of a vector that is used,for example, to transform a cell.

[0160] Alternatively, such recombinant nucleic acids may be part of aviral vector, e.g., based on a vaccinia virus, that could be use tovaccinate a mammal wherein the recombinant nucleic acid is expressed,inducing a protective immunological response in the mammal.

[0161] A “regulatory element” refers to a nucleic acid sequence usuallyderived from untranslated regions of a gene and includes enhancers,promoters, introns, and 5′ and 3′ untranslated regions (UTRs).Regulatory elements interact with host or viral proteins which controltranscription, translation, or RNA stability.

[0162] “Reporter molecules” are chemical or biochemical moieties usedfor labeling a nucleic acid, amino acid, or antibody. Reporter moleculesinclude radionuclides; enzymes; fluorescent, chemiluminescent, orchromogenic agents; substrates; cofactors; inhibitors; magneticparticles; and other moieties known in the art.

[0163] An “RNA equivalent,” in reference to a DNA sequence, is composedof the same linear sequence of nucleotides as the reference DNA sequencewith the exception that all occurrences of the nitrogenous base thymineare replaced with uracil, and the sugar backbone is composed of riboseinstead of deoxyribose.

[0164] The term “sample” is used in its broadest sense. A samplesuspected of containing LIPAM, nucleic acids encoding LIPAM, orfragments thereof may comprise a bodily fluid; an extract from a cell,chromosome, organelle, or membrane isolated from a cell; a cell; genomicDNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; atissue print; etc.

[0165] The terms “specific binding” and “specifically binding” refer tothat interaction between a protein or peptide and an agonist, anantibody, an antagonist, a small molecule, or any natural or syntheticbinding composition. The interaction is dependent upon the presence of aparticular structure of the protein, e.g., the antigenic determinant orepitope, recognized by the binding molecule. For example, if an antibodyis specific for epitope “A,” the presence of a polypeptide comprisingthe epitope A, or the presence of free unlabeled A, in a reactioncontaining free labeled A and the antibody will reduce the amount oflabeled A that binds to the antibody.

[0166] The term “substantially purified” refers to nucleic acid or aminoacid sequences that are removed from their natural environment and areisolated or separated, and are at least 60% free, preferably at least75% free, and most preferably at least 90% free from other componentswith which they are naturally associated.

[0167] A “substitution” refers to the replacement of one or more aminoacid residues or nucleotides by different amino acid residues ornucleotides, respectively.

[0168] “Substrate” refers to any suitable rigid or semi-rigid supportincluding membranes, filters, chips, slides, wafers, fibers, magnetic ornonmagnetic beads, gels, tubing, plates, polymers, microparticles andcapillaries. The substrate can have a variety of surface forms, such aswells, trenches, pins, channels and pores, to which polynucleotides orpolypeptides are bound.

[0169] A “transcript image” or “expression profile” refers to thecollective pattern of gene expression by a particular cell type ortissue under given conditions at a given time.

[0170] “Transformation” describes a process by which exogenous DNA isintroduced into a recipient cell. Transformation may occur under naturalor artificial conditions according to various methods well known in theart, and may rely on any known method for the insertion of foreignnucleic acid sequences into a prokaryotic or eukaryotic host cell. Themethod for transformation is selected based on the type of host cellbeing transformed and may include, but is not limited to, bacteriophageor viral infection, electroporation, heat shock, lipofection, andparticle bombardment. The term “transformed cells” includes stablytransformed cells in which the inserted DNA is capable of replicationeither as an autonomously replicating plasmid or as part of the hostchromosome, as well as transiently transformed cells which express theinserted DNA or RNA for limited periods of time.

[0171] A “transgenic organism,” as used herein, is any organism,including but not limited to animals and plants, in which one or more ofthe cells of the organism contains heterologous nucleic acid introducedby way of human intervention, such as by transgenic techniques wellknown in the art. The nucleic acid is introduced into the cell, directlyor indirectly by introduction into a precursor of the cell, by way ofdeliberate genetic manipulation, such as by microinjection or byinfection with a recombinant virus. The term genetic manipulation doesnot include classical cross-breeding, or in vitro fertilization, butrather is directed to the introduction of a recombinant DNA molecule.The transgenic organisms contemplated in accordance with the presentinvention include bacteria, cyanobacteria, fungi, plants and animals.The isolated DNA of the present invention can be introduced into thehost by methods known in the art, for example infection, transfection,transformation or transconjugation. Techniques for transferring the DNAof the present invention into such organisms are widely known andprovided in references such as Sambrook et al. (1989), supra.

[0172] A “variant” of a particular nucleic acid sequence is defined as anucleic acid sequence having at least 40% sequence identity to theparticular nucleic acid sequence over a certain length of one of thenucleic acid sequences using blastn with the “BLAST 2 Sequences” toolVersion 2.0.9 (May 07, 1999) set at default parameters. Such a pair ofnucleic acids may show, for example, at least 50%, at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% or greater sequence identityover a certain defined length. A variant may be described as, forexample, an “allelic” (as defined above), “splice,” “species,” or“polymorphic” variant. A splice variant may have significant identity toa reference molecule, but will generally have a greater or lesser numberof polynucleotides due to alternate splicing of exons during mRNAprocessing. The corresponding polypeptide may possess additionalfunctional domains or lack domains that are present in the referencemolecule. Species variants are polynucleotide sequences that vary fromone species to another. The resulting polypeptides will generally havesignificant amino acid identity relative to each other. A polymorphicvariant is a variation in the polynucleotide sequence of a particulargene between individuals of a given species. Polymorphic variants alsomay encompass “single nucleotide polymorphisms” (SNPs) in which thepolynucleotide sequence varies by one nucleotide base. The presence ofSNPs may be indicative of, for example, a certain population, a diseasestate, or a propensity for a disease state.

[0173] A “variant” of a particular polypeptide sequence is defined as apolypeptide sequence having at least 40% sequence identity to theparticular polypeptide sequence over a certain length of one of thepolypeptide sequences using blastp with the “BLAST 2 Sequences” toolVersion 2.0.9 (May 07, 1999) set at default parameters. Such a pair ofpolypeptides may show, for example, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% or greater sequence identity over a certain definedlength of one of the polypeptides.

[0174] The Invention

[0175] The invention is based on the discovery of new humanlipid-associated molecules (LIPAM), the polynucleotides encoding LIPAM,and the use of these compositions for the diagnosis, treatment, orprevention of cancers, neurological, autoimmune/inflammatory,gastrointestinal, and cardiovascular disorders, and disorders of lipidmetabolism.

[0176] Table 1 summarizes the nomenclature for the full lengthpolynucleotide and polypeptide sequences of the invention. Eachpolynucleotide and its corresponding polypeptide are correlated to asingle Incyte project identification number (Incyte Project ID). Eachpolypeptide sequence is denoted by both a polypeptide sequenceidentification number (Polypeptide SEQ ID NO:) and an Incyte polypeptidesequence number (Incyte Polypeptide ID) as shown. Each polynucleotidesequence is denoted by both a polynucleotide sequence identificationnumber (Polynucleotide SEQ ID NO:) and an Incyte polynucleotideconsensus sequence number (Incyte Polynucleotide ID) as shown.

[0177] Table 2 shows sequences with homology to the polypeptides of theinvention as identified by BLAST analysis against the GenBank protein(genpept) database. Columns 1 and 2 show the polypeptide sequencidentification number (Polypeptide SEQ ID NO:) and the correspondingIncyte polypeptide sequence number (Incyte Polypeptide ID) forpolypeptides of the invention. Column 3 shows the GenBank identificationnumber (GenBank ID NO:) of the nearest GenBank homolog. Column 4 showsthe probability scores for the matches between each polypeptide and itshomolog(s). Column 5 shows the annotation of the GenBank homolog(s)along with relevant citations where applicable, all of which areexpressly incorporated by reference herein.

[0178] Table 3 shows various structural features of the polypeptides ofthe invention. Columns 1 and 2 show the polypeptide sequenceidentification number (SEQ ID NO:) and the corresponding Incytepolypeptide sequence number (Incyte Polypeptide ID) for each polypeptideof the invention. Column 3 shows the number of amino acid residues ineach polypeptide. Column 4 shows potential phosphorylation sites, andcolumn 5 shows potential glycosylation sites, as determined by theMOTIFS program of the GCG sequence analysis software package (GeneticsComputer Group, Madison Wis.). Column 6 shows amino acid residuescomprising signature sequences, domains, and motifs. Column 7 showsanalytical methods for protein structure/function analysis and in somecases, searchable databases to which the analytical methods wereapplied.

[0179] Together, Tables 2 and 3 summarize the properties of polypeptidesof the invention, and these properties establish that the claimedpolypeptides are lipid-associated molecules. For example, SEQ ID NO:1 is43% identical, from residue M336 to residue R989, to human cytosolicphospholipase A2 beta (cPLA2beta) (GenBank ID g4886978) as determined bythe Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLASTprobability score is 1.6e-161, which indicates the probability ofobtaining the observed polypeptide sequence alignment by chance. In analternative example, SEQ ID NO:3 is 41% identical, from residue M1 toresidue Y644, to rat phospholipase C delta-4 (GenBank ID g4894788) asdetermined by BLAST, with a probability score of 2.5e-126. (See Table2.) SEQ ID NO:3 also contains phosphatidylinositol-specificphospholipase C, X and Y domains and a C2 domain as determined bysearching for statistically significant matches in the hidden Markovmodel (HMM)-based PFAM database of conserved protein family domains.(See Table 3.) Data from BLIMPS analyses provide further corroborativeevidence that SEQ ID NO:3 is a phospholipase C. In an alternativeexample, SEQ ID NO:5 is 40% identical, from residue M1 to residue N316,to human phosphatidylserine-specific phospholipase A1 (GenBank IDg4090960) as determined by BLAST, with a probability score of 3.1e-63.(See Table 2.) SEQ ID NO:5 also contains a lipase domain as determinedby searching for statistically significant matches in the HMM-based PFAMdatabase. (See Table 3.) Data from BLIMPS analyses provide furthercorroborative evidence that SEQ ID NO:5 is a lipase. In an alternativeexample, SEQ ID NO:6 is 45% identical from residue C41 to residue I491,39% identical from residue K582 to residue K899, 33% identical fromresidue S541 to residue G603, and 22% identical from residue S519 toresidue L571, to Mus musculus phospholipase C-L2 (GenBank ID g6705987)as determined by BLAST, with probability scores of 1.1e-164 from residueC41 to residue I491, 1.1e-164 from residue K582 to residue K899, 1.0e-56from residue S541 to residue G603, and 1.1e-55 from residue S519 toresidue L571. (See Table 2.) SEQ ID NO:6 also containsphosphatidylinositol-specific phospholipase active site domains asdetermined by searching for statistically significant matches in theHMM-based PFAM database. (See Table 3.) Data from BLIMPS and MOTIFSanalyses provide further corroborative evidence that SEQ ID NO:6 is aphospholipase. In an alternative example, SEQ ID NO:7 is 90% identical,from residue M1 to residue K1294, to Mus musculus M-RdgB2 retinaldegeneration protein B subtype 2 (GenBank ID g5771350) as determined byBLAST, with a probability score of 0.0. (See Table 2.) SEQ ID NO:7 alsocontains a phosphatidylinositol transfer protein domain as determined bysearching for statistically significant matches in the HMM-based PFAMdatabase. (See Table 3.) Data from BLIMPS, MOTIFS, and additional BLASTanalyses provide further corroborative evidence that SEQ ID NO:7 is aphosphatidylinositol transfer protein. In an alternative example, SEQ IDNO:9 is 81% identical from residue R387 to residue T546, 69% identicalfrom residue T181 to residue Q358, and 61% identical from residue A2 toresidue D191, to Mus musculus TAGL-beta (GenBank ID g6651241) asdetermined by BLAST, with a probability score of 3.8e-188. (See Table2.) Data from additional BLAST analyses provide further corroborativeevidence that SEQ ID NO:9 is a protein peptidoglycan recognitionprecursor. SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:8 were analyzed andannotated in a similar manner. The algorithms and parameters for theanalysis of SEQ ID NO:1-9 are described in Table 7.

[0180] As shown in Table 4, the full length polynucleotide sequences ofthe present invention were assembled using cDNA sequences or coding(exon) sequences derived from genomic DNA, or any combination of thesetwo types of sequences. Column 1 lists the polynucleotide sequenceidentification number (Polynucleotide SEQ ID NO:), the correspondingIncyte polynucleotide consensus sequence number (Incyte ID) for eachpolynucleotide of the invention, and the length of each polynucleotidesequence in basepairs. Column 2 shows the nucleotide start (5′) and stop(3′) positions of the cDNA and/or genomic sequences used to assemble thefull length polynucleotide sequences of the invention, and of fragmentsof the polynucleotide sequences which are useful, for example, inhybridization or amplification technologies that identify SEQ IDNO:10-18 or that distinguish between SEQ ID NO:10-18 and relatedpolynucleotide sequences.

[0181] The polynucleotide fragments described in Column 2 of Table 4 mayrefer specifically, for example, to Incyte cDNAs derived fromtissue-specific cDNA libraries or from pooled cDNA libraries.Alternatively, the polynucleotide fragments described in column 2 mayrefer to GenBank cDNAs or ESTs which contributed to the assembly of thefull length polynucleotide sequences. In addition, the polynucleotidefragments described in column 2 may identify sequences derived from thENSEMBL (The Sanger Centre, Cambridge, UK) database (ie., thosesequences including the designation “ENST”). Alternatively, thepolynucleotide fragments described in column 2 may be derived from theNCBI RefSeq Nucleotide Sequence Records Database (ie., those sequencesincluding the designation “NM” or “NT”) or the NCBI RefSeq ProteinSequence Records (i.e., those sequences including the designation “NP”).Alternatively, the polynucleotide fragments described in column 2 mayrefer to assemblages of both cDNA and Genscan-predicted exons broughttogether by an “exon stitching” algorithm. For example, a polynucleotidesequence identified as FL_XXXXXX_N_(1—)N_(2—)YYYYY_N_(3—)N₄ represents a“stitched” sequence in which XXXXXX is the identification number of thecluster of sequences to which the algorithm was applied, and YYYYY isthe number of the prediction generated by the algorithm, andN_(1, 2, 3 . . .) , if present, represent specific exons that may havebeen manually edited during analysis (See Example V). Alternatively, thepolynucleonide fragments in column 2 may refer to assemblages of exonsbrought together by an “exon-stretching” algorithm. For example, apolynucleotide sequence identified as FLXXXXXX_gAAAAA_gBBBBB_(—)1_N is a“stretched” sequence, with XXXXXX being the Incyte projectidentification number, gAAAAA being the GenBank identification number ofthe human genomic sequence to which the “exon-stretching” algorithm wasapplied, gBBBBB being the GenBank identification number or NCBI RefSeqidentification number of the nearest GenBank protein homolog, and Nreferring to specific exons (See Example V). In instances where a RefSeqsequence was used as a protein homolog for the “exon-stretching”algorithm, a RefSeq identifier (denoted by “NM,” “NP,” or “NT”) may beused in place of the GenBank identifier (i.e., gBBBBB).

[0182] Alternatively, a prefix identifies component sequences that werehand-edited, predicted from genomic DNA sequences, or derived from acombination of sequence analysis methods. The following Table listsexamples of component sequence prefixes and corresponding sequenceanalysis methods associated with the prefixes (see Example IV andExample V). Prefix Type of analysis and/or examples of programs GNN,Exon prediction from genomic sequences using, for example, GFG, GENSCAN(Stanford University, CA, USA) or FGENES ENST (Computer Genomics Group,The Sanger Centre, Cambridge, UK). GBI Hand-edited analysis of genomicsequences. FL Stitched or stretched genomic sequences (see Example V).INCY Full length transcript and exon prediction from mapping of ESTsequences to the genome. Genomic location and EST composition data arecombined to predict the exons and resulting transcript.

[0183] In some cases, Incyte cDNA coverage redundant with the sequencecoverage shown in Table 4 was obtained to confirm the final consensuspolynucleotide sequence, but the relevant Incyte cDNA identificationnumbers are not shown.

[0184] Table 5 shows the representative cDNA libraries for those fulllength polynucleotide sequences which were assembled using Incyte cDNAsequences. The representative cDNA library is the Incyte cDNA librarywhich is most frequently represented by the Incyte cDNA sequences whichwere used to assemble and confirm the above polynucleotide sequences.The tissues and vectors which were used to construct the cDNA librariesshown in Table 5 are described in Table 6.

[0185] The invention also encompasses LIPAM variants. A preferred LIPAMvariant is one which has at least about 80%, or alternatively at leastabout 90%, or even at least about 95% amino acid sequence identity tothe LIPAM amino acid sequence, and which contains at least onefunctional or structural characteristic of LIPAM.

[0186] The invention also encompasses polynucleotides which encodeLIPAM. In a particular embodiment, the invention encompasses apolynucleotide sequence comprising a sequence selected from the groupconsisting of SEQ ID NO:10-18, which encodes LIPAM. The polynucleotidesequences of SEQ ID NO:10-18, as presented in the Sequence Listing,embrace the equivalent RNA sequences, wherein occurrences of thenitrogenous base thymine are replaced with uracil, and the sugarbackbone is composed of ribose instead of deoxyribose.

[0187] The invention also encompasses a variant of a polynucleotidesequence encoding LIPAM. In particular, such a variant polynucleotidesequence will have at least about 70%, or alternatively at least about85%, or even at least about 95% polynucleotide sequence identity to thepolynucleotide sequence encoding LIPAM. A particular aspect of theinvention encompasses a variant of a polynucleotide sequence comprisinga sequence selected from the group consisting of SEQ ID NO:10-18 whichhas at least about 70%, or alternatively at least about 85%, or even atleast about 95% polynucleotide sequence identity to a nucleic acidsequence selected from the group consisting of SEQ ID NO:10-18. Any oneof the polynucleotide variants described above can encode an amino acidsequence which contains at least one functional or structuralcharacteristic of LIPAM.

[0188] In addition, or in the alternative, a polynucleotide variant ofthe invention is a splice variant of a polynucleotide sequence encodingLIPAM. A splice variant may have portions which have significantsequence identity to the polynucleotide sequence encoding LIPAM, butwill generally have a greater or lesser number of polynucleotides due toadditions or deletions of blocks of sequence arising from alternatesplicing of exons during mRNA processing. A splice variant may have lessthan about 70%, or alternatively less than about 60%, or alternativelyless than about 50% polynucleotide sequence identity to thepolynucleotide sequence encoding LIPAM over its entire length; however,portions of the splice variant will have at least about 70%, oralternatively at least about 85%, or alternatively at least about 95%,or alternatively 100% polynucleotide sequence identity to portions ofthe polynucleotide sequence encoding LIPAM. Any one of the splicevariants described above can encode an amino acid sequence whichcontains at least one functional or structural characteristic of LIPAM.

[0189] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude ofpolynucleotide sequences encoding LIPAM, some bearing minimal similarityto the polynucleotide sequences of any known and naturally occurringgene, may be produced. Thus, the invention contemplates each and everypossible variation of polynucleotide sequence that could be made byselecting combinations based on possible codon choices. Thesecombinations are made in accordance with the standard triplet geneticcode as applied to the polynucleotide sequence of naturally occurringLIPAM, and all such variations are to be considered as beingspecifically disclosed.

[0190] Although nucleotide sequences which encode LIPAM and its variantsare generally capable of hybridizing to the nucleotide sequence of thenaturally occurring LIPAM under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding LIPAM or its derivatives possessing a substantially differentcodon usage, e.g., inclusion of non-naturally occurring codons. Codonsmay be selected to increase the rate at which expression of the peptideoccurs in a particular prokaryotic or eukaryotic host in accordance withthe frequency with which particular codons are utilized by the host.Other reasons for substantially altering the nucleotide sequenceencoding LIPAM and its derivatives without altering the encoded aminoacid sequences include the production of RNA transcripts having moredesirable properties, such as a greater half-life, than transcriptsproduced from the naturally occurring sequence.

[0191] The invention also encompasses production of DNA sequences whichencode LIPAM and LIPAM derivatives, or fragments thereof, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents well known in the art. Moreover, syntheticchemistry may be used to introduce mutations into a sequence encodingLIPAM or any fragment thereof.

[0192] Also encompassed by the invention are polynucleotide sequencesthat are capable of hybridizing to the claimed polynucleotide sequences,and, in particular, to those shown in SEQ ID NO:10-18 and fragmentsthereof under various conditions of stringency. (See, e.g., Wahl, G. M.and S. L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A. R.(1987) Methods Enzymol. 152:507-511.) Hybridization conditions,including annealing and wash conditions, are described in “Definitions.”

[0193] Methods for DNA sequencing are well known in the art and may beused to practice any of the embodiments of the invention. The methodsmay employ such enzymes as the Klenow fragment of DNA polymerase I,SEQUENASE (US Biochemical, Cleveland Ohio), Taq polymerase (AppliedBiosystems), thermostable T7 polymerase (Amersham Pharmacia Biotech,Piscataway N.J.), or combinations of polymerases and proofreadingexonucleases such as those found in the ELONGASE amplification system(Life Technologies, Gaithersburg Md.). Preferably, sequence preparationis automated with machines such as the MICROLAB 2200 liquid transfersystem (Hamilton, Reno Nev.), PTC200 thermal cycler (MJ Research,Watertown Mass.) and ABI CATALYST 800 thermal cycler (AppliedBiosystems). Sequencing is then carried out using either the ABI 373 or377 DNA sequencing system (Applied Biosystems), the MEGABACE 1000 DNAsequencing system (Molecular Dynamics, Sunnyvale Calif.), or othersystems known in the art. The resulting sequences are analyzed using avariety of algorithms which are well known in the art. (See, e.g.,Ausubel, F. M. (1997) Short Protocols in Molecular Biology, John Wiley &Sons, New York N.Y., unit 7.7; Meyers, R. A. (1995) Molecular Biologyand Biotechnology, Wiley VCH, New York N.Y., pp. 856-853.)

[0194] The nucleic acid sequences encoding LIPAM may be extendedutilizing a partial nucleotide sequence and employing various PCR-basedmethods known in the art to detect upstream sequences, such as promotersand regulatory elements. For example, one method which may be employed,restriction-site PCR, uses universal and nested primers to amplifyunknown sequence from genomic DNA within a cloning vector. (See, e.g.,Sarkar, G. (1993) PCR Methods Applic. 2:318-322.) Another method,inverse PCR, uses primers that extend in divergent directions to amplifyunknown sequence from a circularized template. The template is derivedfrom restriction fragments comprising a known genomic locus andsurrounding sequences. (See, e.g., Triglia, T. et al. (1988) NucleicAcids Res. 16:8186.) A third method, capture PCR, involves PCRamplification of DNA fragments adjacent to known sequences in human andyeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al.(1991) PCR Methods Applic. 1:111-119.) In this method, multiplerestriction enzyme digestions and ligations may be used to insert anengineered double-stranded sequence into a region of unknown sequencebefore performing PCR. Other methods which may be used to retrieveunknown sequences are known in the art. (See, e.g., Parker, J. D. et al.(1991) Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR,nested primers, and PROMOTERFINDER libraries (Clontech, Palo AltoCalif.) to walk genomic DNA. This procedure avoids the need to screenlibraries and is useful in finding intron/exon junctions. For allPCR-based methods, primers may be designed using commercially availablesoftware, such as OLIGO 4.06 primer analysis software (NationalBiosciences, Plymouth Minn.) or another appropriate program, to be about22 to 30 nucleotides in length, to have a GC content of about 50% ormore, and to anneal to the template at temperatures of about 68° C. to72° C.

[0195] When screening for full length cDNAs, it is preferable to uselibraries that have been size-selected to include larger cDNAs. Inaddition, random-primed libraries, which often include sequencescontaining the 5′ regions of genes, are preferable for situations inwhich an oligo d(T) library does not yield a full-length cDNA. Genomiclibraries may be useful for extension of sequence into 5′non-transcribed regulatory regions.

[0196] Capillary electrophoresis systems which are commerciallyavailable may be used to analyze the size or confirm the nucleotidesequence of sequencing or PCR products. In particular, capillarysequencing may employ flowable polymers for electrophoretic separation,four different nucleotide-specific, laser-stimulated fluorescent dyes,and a charge coupled device camera for detection of the emittedwavelengths. Output/light intensity may be converted to electricalsignal using appropriate software (e.g., GENOTYPER and SEQUENCENAVIGATOR, Applied Biosystems), and the entire process from loading ofsamples to computer analysis and electronic data display may be computercontrolled. Capillary electrophoresis is especially preferable forsequencing small DNA fragments which may be present in limited amountsin a particular sample.

[0197] In another embodiment of the invention, polynucleotide sequencesor fragments thereof which encode LIPAM may be cloned in recombinant DNAmolecules that direct expression of LIPAM, or fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and used to express LIPAM.

[0198] The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterLIPAM-encoding sequences for a variety of purposes including, but notlimited to, modification of the cloning, processing, and/or expressionof the gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example,oligonucleotide-mediated site-directed mutagenesis may be used tointroduce mutations that create new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, and so forth.

[0199] The nucleotides of the present invention may be subjected to DNAshuffling techniques such as MOLECULARBREEDING (Maxygen Inc., SantaClara Calif.; described in U.S. Pat. No. 5,837,458; Chang, C.-C. et al.(1999) Nat. Biotechnol. 17:793-797; Christians, F. C. et al. (1999) Nat.Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol.14:315-319) to alter or improve the biological properties of LIPAM, suchas its biological or enzymatic activity or its ability to bind to othermolecules or compounds. DNA shuffling is a process by which a library ofgene variants is produced using PCR-mediated recombination of genefragments. The library is then subjected to selection or screeningprocedures that identify those gene variants with the desiredproperties. These preferred variants may then be pooled and furthersubjected to recursive rounds of DNA shuffling and selection/screening.Thus, genetic diversity is created through “artificial” breeding andrapid molecular evolution. For example, fragments of a single genecontaining random point mutations may be recombined, screened, and thenreshuffled until the desired properties are optimized. Alternatively,fragments of a given gene may be recombined with fragments of homologousgenes in the same gene family, either from the same or differentspecies, thereby maximizing the genetic diversity of multiple naturallyoccurring genes in a directed and controllable manner.

[0200] In another embodiment, sequences encoding LIPAM may besynthesized, in whole or in part, using chemical methods well known inthe art. (See, e.g., Caruthers, M. H. et al. (1980) Nucleic Acids Symp.Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser.7:225-232.) Alternatively, LIPAM itself or a fragment thereof may besynthesized using chemical methods. For example, peptide synthesis canbe performed using various solution-phase or solid-phase techniques.(See, e.g., Creighton, T. (1984) Proteins, Structures and MolecularProperties, W H Freeman, New York N.Y., pp. 55-60; and Roberge, J. Y. etal. (1995) Science 269:202-204.) Automated synthesis may be achievedusing the ABI 431A peptide synthesizer (Applied Biosystems).Additionally, the amino acid sequence of LIPAM, or any part thereof, maybe altered during direct synthesis and/or combined with sequences fromother proteins, or any part thereof, to produce a variant polypeptide ora polypeptide having a sequence of a naturally occurring polypeptide.

[0201] The peptide may be substantially purified by preparative highperformance liquid chromatography. (See, e.g., Chiez, R. M. and F. Z.Regnier (1990) Methods Enzymol. 182:392-421.) The composition of thesynthetic peptides may be confirmed by amino acid analysis or bysequencing. (See, e.g., Creighton, supra, pp. 28-53.)

[0202] In order to express a biologically active LIPAM, the nucleotidesequences encoding LIPAM or derivatives thereof may be inserted into anappropriate expression vector, i.e., a vector which contains thenecessary elements for transcriptional and translational control of theinserted coding sequence in a suitable host. These elements includeregulatory sequences, such as enhancers, constitutive and induciblepromoters, and 5′ and 3′ untranslated regions in the vector and inpolynucleotide sequences encoding LIPAM. Such elements may vary in theirstrength and specificity. Specific initiation signals may also be usedto achieve more efficient translation of sequences encoding LIPAM. Suchsignals include the ATG initiation codon and adjacent sequences, e.g.the Kozak sequence. In cases where sequences encoding LIPAM and itsinitiation codon and upstream regulatory sequences are inserted into theappropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a fragment thereof, is inserted, exogenoustranslational control signals including an in-frame ATG initiation codonshould be provided by the vector. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers appropriate for the particular host cell system used. (See,e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)

[0203] Methods which are well known to those skilled in the art may beused to construct expression vectors containing sequences encoding LIPAMand appropriate transcriptional and translational control elements.These methods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. (See, e.g., Sambrook, J.et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, Plainview N.Y., ch. 4, 8, and 16-17; Ausubel, F. M. et al. (1995)Current Protocols in Molecular Biology, John Wiley & Sons, New YorkN.Y., ch. 9, 13, and 16.)

[0204] A variety of expression vector/host systems may be utilized tocontain and express sequences encoding LIPAM. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith viral expression vectors (e.g., baculovirus); plant cell systemstransformed with viral expression vectors (e.g., cauliflower mosaicvirus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. (See,e.g., Sambrook, supra; Ausubel, supra; Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509; Engelhard, E. K. et al. (1994)Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum.Gene Ther. 7:1937-1945; Takamatsu, N. (1987) EMBO J. 6:307-311; TheMcGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, NewYork N.Y., pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad.Sci. USA 81:3655-3659; and Harrington, J. J. et al. (1997) Nat. Genet.15:345-355.) Expression vectors derived from retroviruses, adenoviruses,or herpes or vaccinia viruses, or from various bacterial plasmids, maybe used for delivery of nucleotide sequences to the targeted organ,tissue, or cell population. (See, e.g., Di Nicola, M. et al. (1998)Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993) Proc. Natl. Acad.Sci. USA 90(13):6340-6344; Buller, R. M. et al. (1985) Nature317(6040):813-815; McGregor, D. P. et al.(1994) Mol. Immunol.31(3):219-226; and Verma, I. M. and N. Somia (1997) Nature 389:239-242.)The invention is not limited by the host cell employed.

[0205] In bacterial systems, a number of cloning and expression vectorsmay be selected depending upon the use intended for polynucleotidesequences encoding LIPAM. For example, routine cloning, subcloning, andpropagation of polynucleotide sequences encoding LIPAM can be achievedusing a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene,La Jolla Calif.) or PSPORT1 plasmid (Life Technologies). Ligation ofsequences encoding LIPAM into the vector's multiple cloning sitedisrupts the lacZ gene, allowing a colorimetric screening procedure foridentification of transformed bacteria containing recombinant molecules.In addition, these vectors may be useful for in vitro transcription,dideoxy sequencing, single strand rescue with helper phage, and creationof nested deletions in the cloned sequence. (See, e.g., Van Heeke, G.and S. M. Schuster (1989) J. Biol. Chem. 264:5503-5509.) When largequantities of LIPAM are needed, e.g. for the production of antibodies,vectors which direct high level expression of LIPAM may be used. Forexample, vectors containing the strong, inducible SP6 or T7bacteriophage promoter may be used.

[0206] Yeast expression systems may be used for production of LIPAM. Anumber of vectors containing constitutive or inducible promoters, suchas alpha factor, alcohol oxidase, and PGH promoters, may be used in theyeast Saccharomyces cerevisiae or Pichia Rastoris. In addition, suchvectors direct either the secretion or intracellular retention ofexpressed proteins and enable integration of foreign sequences into thehost genome for stable propagation. (See, e.g., Ausubel, 1995, supra;Bitter, G. A. et al. (1987) Methods Enzymol. 153:516-544; and Scorer, C.A. et al. (1994) Bio/Technology 12:181-184.)

[0207] Plant systems may also be used for expression of LIPAM.Transcription of sequences encoding LIPAM may be driven by viralpromoters, e.g., the 35S and 19S promoters of CaMV used alone or incombination with the omega leader sequence from TMV (akamatsu, N. (1987)EMBO J. 6:307-311). Alternatively, plant promoters such as the smallsubunit of RUBISCO or heat shock promoters may be used. (See, e.g.,Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984)Science 224:838-843; and Winter, J. et al. (1991) Results Probl. CellDiffer. 17:85-105.) These constructs can be introduced into plant cellsby direct DNA transformation or pathogen-mediated transfection. (See,e.g., The McGraw Hill Yearbook of Science and Technology (1992) McGrawHill, New York N.Y., pp. 191-196.)

[0208] In mammalian cells, a number of viral-based expression systemsmay be utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding LIPAM may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain infective virus whichexpresses LIPAM in host cells. (See, e.g., Logan, J. and T. Shenk (1984)Proc. Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcriptionenhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used toincrease expression in mammalian host cells. SV40 or EBV-based vectorsmay also be used for high-level protein expression.

[0209] Human artificial chromosomes (HACs) may also be employed todeliver larger fragments of DNA than can be contained in and expressedfrom a plasmid. HACs of about 6 kb to 10 Mb are constructed anddelivered via conventional delivery methods (liposomes, polycationicamino polymers, or vesicles) for therapeutic purposes. (See, e.g.,Harrington, J. J. et al. (1997) Nat. Genet. 15:345-355.)

[0210] For long term production of recombinant proteins in mammaliansystems, stable expression of LIPAM in cell lines is preferred. Forexample, sequences encoding LIPAM can be transformed into cell linesusing expression vectors which may contain viral origins of replicationand/or endogenous expression elements and a selectable marker gene onthe same or on a separate vector. Following the introduction of thevector, cells may be allowed to grow for about 1 to 2 days in enrichedmedia before being switched to selective media. The purpose of theselectable marker is to confer resistance to a selective agent, and itspresence allows growth and recovery of cells which successfully expressthe introduced sequences. Resistant clones of stably transformed cellsmay be propagated using tissue culture techniques appropriate to thecell type.

[0211] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase and adeninephosphoribosyltransferase genes, for use in tk⁻ and apr⁻ cells,respectively. (See, e.g., Wigler, M. et al. (1977) Cell 11:223-232;Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite,antibiotic, or herbicide resistance can be used as the basis forselection. For example, dhfr confers resistance to methotrexate; neoconfers resistance to the aminoglycosides neomycin and G418; and als andpat confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively. (See, e.g., Wigler, M. et al. (1980)Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F. et al.(1981) J. Mol. Biol. 150:1-14.) Additional selectable genes have beendescribed, e.g., trpB and hisD, which alter cellular requirements formetabolites. (Se, e.g., Hartman, S. C. and R. C. Mulligan (1988) Proc.Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins,green fluorescent proteins (GFP; Clontech), β glucuronidase and itssubstrate β-glucuronide, or luciferase and its substrate luciferin maybe used. These markers can be used not only to identify transformants,but also to quantify the amount of transient or stable proteinexpression attributable to a specific vector system. (See, e.g., Rhodes,C. A. (1995) Methods Mol. Biol. 55:121-131.)

[0212] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, the presence and expressionof the gene may need to be confirmed. For example, if the sequenceencoding LIPAM is inserted within a marker gene sequence, transformedcells containing sequences encoding LIPAM can be identified by theabsence of marker gene function. Alternatively, a marker gene can beplaced in tandem with a sequence encoding LIPAM under the control of asingle promoter. Expression of the marker gene in response to inductionor selection usually indicates expression of the tandem gene as well.

[0213] In general, host cells that contain the nucleic acid sequenceencoding LIPAM and that express LIPAM may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCRamplification, and protein bioassay or immunoassay techniques whichinclude membrane, solution, or chip based technologies for the detectionand/or quantification of nucleic acid or protein sequences.

[0214] Immunological methods for detecting and measuring the expressionof LIPAM using either specific polyclonal or monoclonal antibodies areknown in the art. Examples of such techniques include enzyme-linkedimmunosorbent assays (ELISAs), radioimmunoassays (RIAs), andfluorescence activated cell sorting (FACS). A two-site, monoclonal-basedimmunoassay utilizing monoclonal antibodies reactive to twonon-interfering epitopes on LIPAM is preferred, but a competitivebinding assay may be employed. These and other assays are well known inthe art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, aLaboratory Manual, APS Press, St. Paul Minn., Sect. IV; Coligan, J. E.et al. (1997) Current Protocols in Immunology, Greene Pub. Associatesand Wiley-Interscience, New York N.Y.; and Pound, J. D. (1998)Immunochemical Protocols, Humana Press, Totowa N.J.)

[0215] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encoding LIPAMinclude oligolabeling, nick translation, end-labeling, or PCRamplification using a labeled nucleotide. Alternatively, the sequencesencoding LIPAM, or any fragments thereof, may be cloned into a vectorfor the production of an mRNA probe. Such vectors are known in the art,are commercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits, such as those provided byAmersham Pharmacia Biotech, Promega (Madison Wis.), and US Biochemical.Suitable reporter molecules or labels which may be used for ease ofdetection include radionuclides, enzymes, fluorescent, chemiluminescent,or chromogenic agents, as well as substrates, cofactors, inhibitors,magnetic particles, and the like.

[0216] Host cells transformed with nucleotide sequences encoding LIPAMmay be cultured under conditions suitable for the expression andrecovery of the protein from cell culture. The protein produced by atransformed cell may be secreted or retained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing polynucleotides whichencode LIPAM may be designed to contain signal sequences which directsecretion of LIPAM through a prokaryotic or eukaryotic cell membrane.

[0217] In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” or “pro” form ofthe protein may also be used to specify protein targeting, folding,and/or activity. Different host cells which have specific cellularmachinery and characteristic mechanisms for post-translationalactivities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available fromthe American Type Culture Collection (ATCC, Manassas Va.) and may bechosen to ensure the correct modification and processing of the foreignprotein.

[0218] In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding LIPAM may be ligated to aheterologous sequence resulting in translation of a fusion protein inany of the aforementioned host systems. For example, a chimeric LIPAMprotein containing a heterologous moiety that can be recognized by acommercially available antibody may facilitate the screening of peptidelibraries for inhibitors of LIPAM activity. Heterologous protein andpeptide moieties may also facilitate purification of fusion proteinsusing commercially available affinity matrices. Such moieties include,but are not limited to, glutathione S-transferase (GST), maltose bindingprotein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP),6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and6-His enable purification of their cognate fusion proteins onimmobilized glutathione, maltose, phenylarsine oxide, calmodulin, andmetal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA)enable immnunoaffinity purification of fusion proteins usingcommercially available monoclonal and polyclonal antibodies thatspecifically recognize these epitope tags. A fusion protein may also beengineered to contain a proteolytic cleavage site located between theLIPAM encoding sequence and the heterologous protein sequence, so thatLIPAM may be cleaved away from the heterologous moiety followingpurification. Methods for fusion protein expression and purification arediscussed in Ausubel (1995, supra, ch. 10). A variety of commerciallyavailable kits may also be used to facilitate expression andpurification of fusion proteins.

[0219] In a further embodiment of the invention, synthesis ofradiolabeled LIPAM may be achieved in vitro using the TNT rabbitreticulocyte lysate or wheat germ extract system (Promega). Thesesystems couple transcription and translation of protein-coding sequencesoperably associated with the T7, T3, or SP6 promoters. Translation takesplace in the presence of a radiolabeled amino acid precursor, forexample, ³⁵S-methionine.

[0220] LIPAM of the present invention or fragments thereof may be usedto screen for compounds that specifically bind to LIPAM. At least oneand up to a plurality of test compounds may be screened for specificbinding to LIPAM. Examples of test compounds include antibodies,oligonucleotides, proteins (e.g., receptors), or small molecules.

[0221] In one embodiment, the compound thus identified is closelyrelated to the natural ligand of LIPAM, e.g., a ligand or fragmentthereof, a natural substrate, a structural or functional mimetic, or anatural binding partner. (See, e.g., Coligan, J. E. et al. (1991)Current Protocols in Immunology 1(2): Chapter 5.) Similarly, thecompound can be closely related to the natural receptor to which LIPAMbinds, or to at least a fragment of the receptor, e.g., the ligandbinding site. In either case, the compound can be rationally designedusing known techniques. In one embodiment, screening for these compoundsinvolves producing appropriate cells which express LIPAM, either as asecreted protein or on the cell membrane. Preferred cells include cellsfrom mammals, yeast, Drosophila, or E. coli. Cells expressing LIPAM orcell membrane fractions which contain LIPAM are then contacted with atest compound and binding, stimulation, or inhibition of activity ofeither LIPAM or the compound is analyzed.

[0222] An assay may simply test binding of a test compound to thepolypeptide, wherein binding is detected by a fluorophore, radioisotope,enzyme conjugate, or other detectable label. For example, the assay maycomprise the steps of combining at least one test compound with LIPAM,either in solution or affixed to a solid support, and detecting thebinding of LIPAM to the compound. Alternatively, the assay may detect ormeasure binding of a test compound in the presence of a labeledcompetitor. Additionally, the assay may be carried out using cell-freepreparations, chemical libraries, or natural product mixtures, and thetest compound(s) may be free in solution or affixed to a solid support.

[0223] LIPAM of the present invention or fragments thereof may be usedto screen for compounds that modulate the activity of LIPAM. Suchcompounds may include agonists, antagonists, or partial or inverseagonists. In one embodiment, an assay is performed under conditionspermissive for LIPAM activity, wherein LIPAM is combined with at leastone test compound, and the activity of LIPAM in the presence of a testcompound is compared with the activity of LIPAM in the absence of thetest compound. A change in the activity of LIPAM in the presence of thetest compound is indicative of a compound that modulates the activity ofLIPAM. Alternatively, a test compound is combined with an in vitro orcell-free system comprising LIPAM under conditions suitable for LIPAMactivity, and the assay is performed. In either of these assays, a testcompound which modulates the activity of LIPAM may do so indirectly andneed not come in direct contact with the test compound. At least one andup to a plurality of test compounds may be screened.

[0224] In another embodiment, polynucleotides encoding LIPAM or theirmammalian homologs may be “knocked out” in an animal model system usinghomologous recombination in embryonic stem (ES) cells. Such techniquesare well known in the art and are useful for the generation of animalmodels of human disease. (See, e.g., U.S. Pat. No. 5,175,383 and U.S.Pat. No. 5,767,337.) For example, mouse ES cells, such as the mouse129/SvJ cell line, are derived from the early mouse embryo and grown inculture. The ES cells are transformed with a vector containing the geneof interest disrupted by a marker gene, e.g., the neomycinphosphotransferase gene (neo; Capecchi, M. R. (1989) Science244:1288-1292). The vector integrates into the corresponding region ofthe host genome by homologous recombination. Alternatively, homologousrecombination takes place using the Cre-loxP system to knockout a geneof interest in a tissue- or developmental stage-specific manner (Marth,J. D. (1996) Clin. Invest. 97:1999-2002; Wagner, K. U. et al. (1997)Nucleic Acids Res. 25:4323-4330). Transformed ES cells are identifiedand microinjected into mouse cell blastocysts such as those from theC57BL/6 mouse strain. The blastocysts are surgically transferred topseudopregnant dams, and the resulting chimeric progeny are genotypedand bred to produce heterozygous or homozygous strains. Transgenicanimals thus generated may be tested with potential therapeutic or toxicagents.

[0225] Polynucleotides encoding LIPAM may also be manipulated in vitroin ES cells derived from human blastocysts. Human ES cells have thepotential to differentiate into at least eight separate cell lineagesincluding endoderm, mesoderm, and ectodermal cell types. These celllineages differentiate into, for example, neural cells, hematopoieticlineages, and cardiomyocytes (Thomson, J. A. et al. (1998) Science282:1145-1147).

[0226] Polynucleotides encoding LIPAM can also be used to create“knockin” humanized animals (pigs) or transgenic animals (mice or rats)to model human disease. With knockin technology, a region of apolynucleotide encoding LIPAM is injected into animal ES cells, and theinjected sequence integrates into the animal cell genome. Transformedcells are injected into blastulae, and th blastulae are implanted asdescribed above. Transgenic progeny or inbred lines are studied andtreated with potential pharmaceutical agents to obtain information ontreatment of a human disease. Alternatively, a mammal inbred tooverexpress LIPAM, e.g., by secreting LIPAM in its milk, may also serveas a convenient source of that protein (Janne, J. et al. (1998)Biotechnol. Annu. Rev. 4:55-74).

[0227] Therapeutics

[0228] Chemical and structural similarity, e.g., in the context ofsequences and motifs, exists between regions of LIPAM andlipid-associated molecules. In addition, examples of tissues expressingLIPAM are normal lung, cancerous lung, and diseased thyroid tissue, andalso can be found in Table 6. Therefore, LIPAM appears to play a role incancers, neurological, autoimmune/inflammatory, gastrointestinal, andcardiovascular disorders, and disorders of lipid metabolism. In thetreatment of disorders associated with increased LIPAM expression oractivity, it is desirable to decrease the expression or activity ofLIPAM. In the treatment of disorders associated with decreased LIPAMexpression or activity, it is desirable to increase the expression oractivity of LIPAM.

[0229] Therefore, in one embodiment, LIPAM or a fragment or derivativethereof may be administered to a subject to treat or prevent a disorderassociated with decreased expression or activity of LIPAM. Examples ofsuch disorders include, but are not limited to, a cancer, such asadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; a cardiovascular disordersuch as arteriovenous fistula, atherosclerosis, hypertension,vasculitis, Raynaud's disease, aneurysms, arterial dissections, varicoseveins, thrombophlebitis and phlebothrombosis, vascular tumors, andcomplications of thrombolysis, balloon angioplasty, vascularreplacement, and coronary artery bypass graft surgery, congestive heartfailure, ischemic heart disease, angina pectoris, myocardial infarction,hypertensive heart disease, degenerative valvular heart disease,calcific aortic valve stenosis, congenitally bicuspid aortic valve,mitral annular calcification, mitral valve prolapse, rheumatic fever andrheumatic heart disease, infective endocarditis, nonbacterial thromboticendocarditis, endocarditis of systemic lupus erythematosus, carcinoidheart disease, cardiomyopathy, myocarditis, pericarditis, neoplasticheart disease, congenital heart disease, and complications of cardiactransplantation, congenital lung anomalies, atelectasis, pulmonarycongestion and edema, pulmonary embolism, pulmonary hemorrhage,pulmonary infarction, pulmonary hypertension, vascular sclerosis,obstructive pulmonary disease, restrictive pulmonary disease, chronicobstructive pulmonary disease, emphysema, chronic bronchitis, bronchialasthma, bronchiectasis, bacterial pneumonia, viral and mycoplasmalpneumonia, lung abscess, pulmonary tuberculosis, diffuse interstitialdiseases, pneumoconioses, sarcoidosis, idiopathic pulmonary fibrosis,desquamative interstitial pneumonitis, hypersensitivity pneumonitis,pulmonary eosinophilia bronchiolitis obliterans-organizing pneumonia,diffuse pulmonary hemorrhage syndromes, Goodpasture's syndromes,idiopathic pulmonary hemosiderosis, pulmonary involvement incollagen-vascular disorders, pulmonary alveolar proteinosis, lungtumors, inflammatory and noninflammatory pleural effusions,pneumothorax, pleural tumors, drug-induced lung disease,radiation-induced lung disease, and complications of lungtransplantation; a neurological disorder such as epilepsy, ischemiccerebrovascular disease, stroke, cerebral neoplasms, Alzheimer'sdisease, Pick's disease, Huntington's disease, dementia, Parkinson'sdisease and other extrapyramidal disorders, amyotrophic lateralsclerosis and other motor neuron disorders, progressive neural muscularatrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosisand other demyelinating diseases, bacterial and viral meningitis, brainabscess, subdural empyema, epidural abscess, suppurative intracranialthrombophlebitis, myelitis and radiculitis, viral central nervous systemdisease, prion diseases including kuru, Creutzfeldt-Jakob disease, andGerstmann-Straussler-Scheinker syndrome, fatal familial insomnia,nutritional and metabolic diseases of the nervous system,neurofibromatosis, tuberous sclerosis, cerebelloretinalhemangioblastomatosis, encephalotrigeminal syndrome, mental retardationand other developmental disorders of the central nervous systemincluding Down syndrome, cerebral palsy, neuroskeletal disorders,autonomic nervous system disorders, cranial nerve disorders, spinal corddiseases, muscular dystrophy and other neuromuscular disorders,peripheral nervous system disorders, dermatomyositis and polymyositis,inherited, metabolic, endocrine, and toxic myopathies, myastheniagravis, periodic paralysis, mental disorders including mood, anxiety,and schizophrenic disorders, seasonal affective disorder (SAD),akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia,dystonias, paranoid psychoses, postherpetic neuralgia, Tourette'sdisorder, progressive supranuclear palsy, corticobasal degeneration, andfamilial frontotemporal dementia; an autoimmune/inflammatory disordersuch as acquired immunodeficiency syndrome (AIDS), Addison's disease,adult respiratory distress syndrome, allergies, ankylosing spondylitis,amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolyticanemia, autoimmune thyroiditis, autoimmunepolyendocrinopathy-candidiasis-ectodermal dystrophy (APECED),bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopicdermatitis, dermatomyositis, diabetes mellitus, emphysema, episodiclymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythemanodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome,gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,irritable bowel syndrome, multiple sclerosis, myasthenia gravis,myocardial or pericardial inflammation, osteoarthritis, osteoporosis,pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoidarthritis, scleroderma, Sjögren's syndrome, systemic anaphylaxis,systemic lupus erythematosus, systemic sclerosis, thrombocytopenicpurpura, ulcerative colitis, uveitis, Werner syndrome, complications ofcancer, hemodialysis, and extracorporeal circulation, viral, bacterial,fungal, parasitic, protozoal, and helminthic infections, and trauma; agastrointestinal disorder such as dysphagia, peptic esophagitis,esophageal spasm, esophageal stricture, esophageal carcinoma, dyspepsia,indigestion, gastritis, gastric carcinoma, anorexia, nausea, emesis,gastroparesis, antral or pyloric edema, abdominal angina, pyrosis,gastroenteritis, intestinal obstruction, infections of the intestinaltract, peptic ulcer, cholelithiasis, cholecystitis, cholestasis,pancreatitis, pancreatic carcinoma, biliary tract disease, hepatitis,hyperbilirubinemia, cirrhosis, passive congestion of the liver,hepatoma, infectious colitis, ulcerative colitis, ulcerative proctitis,Crohn's disease, Whipple's disease, Mallory-Weiss syndrome, coloniccarcinoma, colonic obstruction, irritable bowel syndrome, short bowelsyndrome, diarrhea, constipation, gastrointestinal hemorrhage, acquiredimmunodeficiency syndrome (AIDS) enteropathy, jaundice, hepaticencephalopathy, hepatorenal syndrome, hepatic steatosis,hemochromatosis, Wilson's disease, alpha,-antitrypsin deficiency, Reye'ssyndrome, primary sclerosing cholangitis, liver infarction, portal veinobstruction and thrombosis, centrilobular necrosis, peliosis hepatis,hepatic vein thrombosis, veno-occlusive disease, preeclampsia,eclampsia, acute fatty liver of pregnancy, intrahepatic cholestasis ofpregnancy, and hepatic tumors including nodular hyperplasias, adenomas,and carcinomas; and a disorder of lipid metabolism such as fatty liver,cholestasis, primary biliary cirrhosis, carnitine deficiency, carnitinepalmitoyltransferase deficiency, myoadenylate deaminase deficiency,hypertriglyceridemia, lipid storage disorders such Fabry's disease,Gaucher's disease, Niemann-Pick's disease, metachromatic leukodystrophy,adrenoleukodystrophy, GM₂ gangliosidosis, and ceroid lipofuscinosis,abetalipoproteinemia, Tangier disease, hyperlipoproteinemia, diabetesmellitus, lipodystrophy, lipomatoses, acute panniculitis, disseminatedfat necrosis, adiposis dolorosa, lipoid adrenal hyperplasia, minimalchange disease, lipomas, atherosclerosis, hypercholesterolemia,hypercholesterolemia with hypertriglyceridemia, primaryhypoalphalipoproteinemia, hypothyroidism, renal disease, liver disease,lecithin:cholesterol acyltransferase deficiency, cerebrotendinousxanthomatosis, sitosterolemia, hypocholesterolemia, Tay-Sachs disease,Sandhoff's disease, hyperlipidemia, hyperlipemia, lipid myopathies, andobesity.

[0230] In another embodiment, a vector capable of expressing LIPAM or afragment or derivative thereof may be administered to a subject to treator prevent a disorder associated with decreased expression or activityof LIPAM including, but not limited to, those described above.

[0231] In a further embodiment, a composition comprising a substantiallypurified LIPAM in conjunction with a suitable pharmaceutical carrier maybe administered to a subject to treat or prevent a disorder associatedwith decreased expression or activity of LIPAM including, but notlimited to, those provided above.

[0232] In still another embodiment, an agonist which modulates theactivity of LIPAM may be administered to a subject to treat or prevent adisorder associated with decreased expression or activity of LIPAMincluding, but not limited to, those listed above.

[0233] In a further embodiment, an antagonist of LIPAM may beadministered to a subject to treat or prevent a disorder associated withincreased expression or activity of LIPAM. Examples of such disordersinclude, but are not limited to, those cancers, neurological,autoimnune/inflammatory, gastrointestinal, and cardiovascular disorders,and disorders of lipid metabolism, described above. In one aspect, anantibody which specifically binds LIPAM may be used directly as anantagonist or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissues which express LIPAM.

[0234] In an additional embodiment, a vector expressing the complementof the polynucleotide encoding LIPAM may be administered to a subject totreat or prevent a disorder associated with increased expression oractivity of LIPAM including, but not limited to, those described above.

[0235] In other embodiments, any of the proteins, antagonists,antibodies, agonists, complementary sequences, or vectors of theinvention may be administered in combination with other appropriatetherapeutic agents. Selection of the appropriate agents for use incombination therapy may be made by one of ordinary skill in the art,according to conventional pharmaceutical principles. The combination oftherapeutic agents may act synergistically to effect the treatment orprevention of the various disorders described above. Using thisapproach, one may be able to achieve therapeutic efficacy with lowerdosages of each agent, thus reducing the potential for adverse sideeffects.

[0236] An antagonist of LIPAM may be produced using methods which aregenerally known in the art. In particular, purified LIPAM may be used toproduce antibodies or to screen libraries of pharmaceutical agents toidentify those which specifically bind LIPAM. Antibodies to LIPAM mayalso be generated using methods that are well known in the art. Suchantibodies may include, but are not limited to, polyclonal, monoclonal,chimeric, and single chain antibodies, Fab fragments, and fragmentsproduced by a Fab expression library. Neutralizing antibodies (i.e.,those which inhibit dimer formation) are generally preferred fortherapeutic use. Single chain antibodies (e.g., from camels or llamas)may be potent enzyme inhibitors and may have advantages in the design ofpeptide mimetics, and in the development of immuno-adsorbents andbiosensors (Muyldermans, S. (2001) J. Biotechnol. 74:277-302).

[0237] For the production of antibodies, various hosts including goats,rabbits, rats, mice, camels, dromedaries, llamas, humans, and others maybe immunized by injection with LIPAM or with any fragment oroligopeptide thereof which has immunogenic properties. Depending on thehost species, various adjuvants may be used to increase immunologicalresponse. Such adjuvants include, but are not limited to, Freund's,mineral gels such as aluminum hydroxide, and surface active substancessuch as lysolecithin, pluronic polyols, polyanions, peptides, oilemulsions, KLH, and dinitrophenol. Among adjuvants used in humans, BCG(bacilli Calmette-Guerin) and Corynebacterium parvum are especiallypreferable.

[0238] It is preferred that the oligopeptides, peptides, or fragmentsused to induce antibodies to LIPAM have an amino acid sequenceconsisting of at least about 5 amino acids, and generally will consistof at least about 10 amino acids. It is also preferable that theseoligopeptides, peptides, or fragments are identical to a portion of theamino acid sequence of the natural protein. Short stretches of LIPAMamino acids may be fused with those of another protein, such as KLH, andantibodies to the chimeric molecule may be produced.

[0239] Monoclonal antibodies to LIPAM may be prepared using anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique. (See, e.g., Kohler, G. et al. (1975) Nature256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42;Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030; andCole, S. P. et al. (1984) Mol. Cell Biol. 62:109-120.)

[0240] In addition, techniques developed for the production of “chimericantibodies,” such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used. (See, e.g., Morrison, S. L. et al.(1984) Proc. Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M. S. et al.(1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature314:452-454.) Alternatively, techniques described for the production ofsingle chain antibodies may be adapted, using methods known in the art,to produce LIPAM-specific single chain antibodies. Antibodies withrelated specificity, but of distinct idiotypic composition, may begenerated by chain shuffling from random combinatorial immunoglobulinlibraries. (See, e.g., Burton, D. R. (1991) Proc. Natl. Acad. Sci. USA88:10134-10137.)

[0241] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening immunoglobulin libraries orpanels of highly specific binding reagents as disclosed in theliterature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci.USA 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)

[0242] Antibody fragments which contain specific binding sites for LIPAMmay also be generated. For example, such fragments include, but are notlimited to, F(ab′)₂ fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab′)2 fragments. Alternatively, Fab expressionlibraries may be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity. (See, e.g., Huse,W. D. et al. (1989) Science 246:1275-1281.)

[0243] Various immunoassays may be used for screening to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificities are well knownin the art. Such immunoassays typically involve the measurement ofcomplex formation between LIPAM and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering LIPAM epitopes is generally used, but a competitivebinding assay may also be employed (Pound, surra).

[0244] Various methods such as Scatchard analysis in conjunction withradioimmunoassay techniques may be used to assess the affinity ofantibodies for LIPAM. Affinity is expressed as an association constant,K_(a), which is defined as the molar concentration of LIPAM-antibodycomplex divided by the molar concentrations of free antigen and freeantibody under equilibrium conditions. The K_(a) determined for apreparation of polyclonal antibodies, which are heterogeneous in theiraffinities for multiple LIPAM epitopes, represents the average affinity,or avidity, of the antibodies for LIPAM. The K_(a) determined for apreparation of monoclonal antibodies, which are monospecific for aparticular LIPAM epitope, represents a true measure of affinity.High-affinity antibody preparations with K_(a) ranging from about 10⁹ to10¹² L/mole are preferred for use in immunoassays in which theLIPAM-antibody complex must withstand rigorous manipulations.Low-affinity antibody preparations with K_(a) ranging from about 10⁶ to10⁷ L/mole are preferred for use in immunopurification and similarprocedures which ultimately require dissociation of LIPAM, preferably inactive form, from the antibody (Catty, D. (1988) Antibodies, Volume I: APractical Approach, IRL Press, Washington D.C.; Liddell, J. E. and A.Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley &Sons, New York N.Y.).

[0245] The titer and avidity of polyclonal antibody preparations may befurther evaluated to determine the quality and suitability of suchpreparations for certain downstream applications. For example, apolyclonal antibody preparation containing at least 1-2 mg specificantibody/ml, preferably 5-10 mg specific antibody/ml, is generallyemployed in procedures requiring precipitation of LIPAM-antibodycomplexes. Procedures for evaluating antibody specificity, titer, andavidity, and guidelines for antibody quality and usage in variousapplications, are generally available. (See, e.g., Catty, supra, andColigan et al. supra.)

[0246] In another embodiment of the invention, the polynucleotidesencoding LIPAM, or any fragment or complement thereof, may be used fortherapeutic purposes. In one aspect, modifications of gene expressioncan be achieved by designing complementary sequences or antisensemolecules (DNA, RNA, PNA, or modified oligonucleotides) to the coding orregulatory regions of the gene encoding LIPAM. Such technology is wellknown in the art, and antisense oligonucleotides or larger fragments canbe designed from various locations along the coding or control regionsof sequences encoding LIPAM. (See, e.g., Agrawal, S., ed. (1996)Antisense Therapeutics, Humana Press Inc., Totawa N.J.)

[0247] In therapeutic use, any gene delivery system suitable forintroduction of the antisense sequences into appropriate target cellscan be used. Antisense sequences can be delivered intracellularly in theform of an expression plasmid which, upon transcription, produces asequence complementary to at least a portion of the cellular sequenceencoding the target protein. (See, e.g., Slater, J. E. et al. (1998) J.Allergy Clin. Immunol. 102(3):469-475; and Scanlon, K. J. et al. (1995)9(13):1288-1296.) Antisense sequences can also be introducedintracellularly through the use of viral vectors, such as retrovirus andadeno-associated virus vectors. (See, e.g., Miller, A. D. (1990) Blood76:271; Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol.Ther. 63(3):323-347.) Other gene delivery mechanisms includeliposome-derived systems, artificial viral envelopes, and other systemsknown in the art. (See, e.g., Rossi, J. J. (1995) Br. Med. Bull.51(1):217-225; Boado, R. J. et al. (1998) J. Pharm. Sci.87(11):1308-1315; and Morris, M. C. et al. (1997) Nucleic Acids Res.25(14):2730-2736.)

[0248] In another embodiment of the invention, polynucleotides encodingLIPAM may be used for somatic or germline gene therapy. Gene therapy maybe performed to (i) correct a genetic deficiency (e.g., in the cases ofsevere combined immunodeficiency (SCID)-X1 disease characterized byX-linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science288:669-672), severe combined immunodeficiency syndrome associated withan inherited adenosine deaminase (ADA) deficiency (Blaese, R. M. et al.(1995) Science 270:475-480; Bordignon, C. et al. (1995) Science270:470-475), cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216;Crystal, R. G. et al. (1995) Hum. Gene Therapy 6:643-666; Crystal, R. G.et al. (1995) Hum. Gene Therapy 6:667-703), thalassamias, familialhypercholesterolemia, and hemophilia resulting from Factor VIII orFactor IX deficiencies (Crystal, R. G. (1995) Science 270:404-410; Vrma, I. M. and N. Somia (1997) Nature 389:239-242)), (ii) express aconditionally lethal gene product (e.g., in the case of cancers whichresult from unregulated cell proliferation), or (iii) express a proteinwhich affords protection against intracellular parasites (e.g., againsthuman retroviruses, such as human immunodeficiency virus (IRV)(Baltimore, D. (1988) Nature 335:395-396; Poeschla, E. et al. (1996)Proc. Natl. Acad. Sci. USA 93:11395-11399), hepatitis B or C virus (HBV,HCV); fungal parasites, such as Candida albicans and Paracoccidioidesbrasiliensis; and protozoan parasites such as Plasmodium falciparum andTrypanosoma cruzi). In the case where a genetic deficiency in LIPAMexpression or regulation causes disease, the expression of LIPAM from anappropriate population of transduced cells may alleviate the clinicalmanifestations caused by the genetic deficiency.

[0249] In a further embodiment of the invention, diseases or disorderscaused by deficiencies in LIPAM are treated by constructing mammalianexpression vectors encoding LIPAM and introducing these vectors bymechanical means into LIPAM-deficient cells. Mechanical transfertechnologies for use with cells in vivo or ex vitro include (i) directDNA microinjection into individual cells, (ii) ballistic gold particledelivery, (iii) liposome-mediated transfection, (iv) receptor-mediatedgene transfer, and (v) the use of DNA transposons (Morgan, R. A. and W.F. Anderson (1993) Annu. Rev. Biochem. 62:191-217; Ivics, Z. (1997) Cell91:501-510; Boulay, J-L. and H. Récipon (1998) Curr. Opin. Biotechnol.9:445450).

[0250] Expression vectors that may be effective for the expression ofLIPAM include, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2,PREP, PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad Calif.),PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla Calif.), andPTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo AltoCalif.). LIPAM may be expressed using (i) a constitutively activepromoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV),SV40 virus, thymidine kinase (TK), or β-actin genes), (ii) an induciblepromoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H.Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al.(1995) Science 268:1766-1769; Rossi, F. M. V. and H. M. Blau (1998)Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REXplasmid (Invitrogen)); the ecdysone-inducible promoter (available in theplasmids PVGRXR and PIND; Invitrogen); the FK506/rapamycin induciblepromoter, or the RU486/mifepristone inducible promoter (Rossi, F. M. V.and H. M. Blau, supra)), or (iii) a tissue-specific promoter or thenative promoter of the endogenous gene encoding LIPAM from a normalindividual.

[0251] Commercially available liposome transformation kits (e.g., thePERFECT LIPID TRANSFECTION KIT, available from Invitrogen) allow onewith ordinary skill in the art to deliver polynucleotides to targetcells in culture and require minimal effort to optimize experimentalparameters. In the alternative, transformation is performed using thecalcium phosphate method (Graham, F. L. and A. J. Eb (1973) Virology52:456-467), or by electroporation (Neumann, E. et al. (1982) EMBO J.1:841-845). The introduction of DNA to primary cells requiresmodification of these standardized mammalian transfection protocols.

[0252] In another embodiment of the invention, diseases or disorderscaused by genetic defects with respect to LIPAM expression are treatedby constructing a retrovirus vector consisting of (i) the polynucleotideencoding LIPAM under the control of an independent promoter or theretrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNApackaging signals, and (iii) a Rev-responsive element (RRE) along withadditional retrovirus cis-acting RNA sequences and coding sequencesrequired for efficient vector propagation. Retrovirus vectors (e.g., PFBand PFBNEO) are commercially available (Stratagene) and are based onpublished data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci. USA92:6733-6737), incorporated by reference herein. The vector ispropagated in an appropriate vector producing cell line (VPCL) thatexpresses an envelope gene with a tropism for receptors on the targetcells or a promiscuous envelope protein such as VSVg (Armentano, D. etal. (1987) J. Virol. 61:1647-1650; Bender, M. A. et al. (1987) J. Virol.61:1639-1646; Adam, M. A. and A. D. Miller (1988) J. Virol.62:3802-3806; Dull, T. et al. (1998) J. Virol. 72:8463-8471; Zufferey,R. et al. (1998) J. Virol. 72:9873-9880). U.S. Pat. No. 5,910,434 toRigg (“Method for obtaining retrovirus packaging cell lines producinghigh transducing efficiency retroviral supernatant”) discloses a methodfor obtaining retrovirus packaging cell lines and is hereby incorporatedby reference. Propagation of retrovirus vectors, transduction of apopulation of cells (e.g., CD4+ T-cells), and the return of transducedcells to a patient are procedures well known to persons skilled in theart of gene therapy and have been well documented (Ranga, U. et al.(1997) J. Virol. 71:7020-7029; Bauer, G. et al. (1997) Blood89:2259-2267; Bonyhadi, M. L. (1997) J. Virol. 71:4707-4716; Ranga, U.et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997)Blood 89:2283-2290).

[0253] In the alternative, an adenovirus-based gene therapy deliverysystem is used to deliver polynucleotides encoding LIPAM to cells whichhave one or more genetic abnormalities with respect to the expression ofLIPAM. The construction and packaging of adenovirus-based vectors arewell known to those with ordinary skill in the art. Replicationdefective adenovirus vectors have proven to be versatile for importinggenes-encoding immunoregulatory proteins into intact islets in thepancreas (Csete, M. E. et al. (1995) Transplantation 27:263-268).Potentially useful adenoviral vectors are described in U.S. Pat. No.5,707,618 to Armentano (“Adenovirus vectors for gene therapy”), herebyincorporated by reference. For adenoviral vectors, see also Antinozzi,P. A. et al. (1999) Annu. Rev. Nutr. 19:511-544 and Verma, I. M. and N.Somia (1997) Nature 18:389:239-242, both incorporated by referenceherein.

[0254] In another alternative, a herpes-based, gene therapy deliverysystem is used to deliver polynucleotides encoding LIPAM to target cellswhich have one or more genetic abnormalities with respect to theexpression of LIPAM. The use of herpes simplex virus (HSV)-based vectorsmay be especially valuable for introducing LIPAM to cells of the centralnervous system, for which HSV has a tropism. The construction andpackaging of herpes-based vectors are well known to those with ordinaryskill in the art. A replication-competent herpes simplex virus (HSV)type 1-based vector has been used to deliver a reporter gene to the eyesof primates (Liu, X. et al. (1999) Exp. Eye Res. 169:385-395). Theconstruction of a HSV-1 virus vector has also been disclosed in detailin U.S. Pat. No. 5,804,413 to DeLuca (“Herpes simplex virus strains forgene transfer”), which is hereby incorporated by reference. U.S. Pat.No. 5,804,413 teaches the use of recombinant HSV d92 which consists of agenome containing at least one exogenous gene to be transferred to acell under the control of the appropriate promoter for purposesincluding human gene therapy. Also taught by this patent are theconstruction and use of recombinant HSV strains deleted for ICP4, ICP27and ICP22. For HSV vectors, see also Goins, W. F. et al. (1999) J.Virol. 73:519-532 and Xu, H. et al. (1994) Dev. Biol. 163:152-161,hereby incorporated by reference. The manipulation of cloned herpesvirussequences, the generation of recombinant virus following thetransfection of multiple plasmids containing different segments of thelarge herpesvirus genomes, the growth and propagation of herpesvirus,and the infection of cells with herpesvirus are techniques well known tothose of ordinary skill in the art.

[0255] In another alternative, an alphavirus (positive, single-strandedRNA virus) vector is used to deliver polynucleotides encoding LIPAM totarget cells. The biology of the prototypic alphavirus, Semliki ForestVirus (SFV), has been studied extensively and gene transfer vectors havebeen based on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin.Biotechnol. 9:464-469). During alphavirus RNA replication, a subgenomicRNA is generated that normally encodes the viral capsid proteins. Thissubgenomic RNA replicates to higher levels than the full length genomicRNA, resulting in the overproduction of capsid proteins relative to theviral proteins with enzymatic activity (e.g., protease and polymerase).Similarly, inserting the coding sequence for LIPAM into the alphavirusgenome in place of the capsid-coding region results in the production ofa large number of LIPAM-coding RNAs and the synthesis of high levels ofLIPAM in vector transduced cells. While alphavirus infection istypically associated with cell lysis within a few days, the ability toestablish a persistent infection in hamster normal kidney cells (BHK-21)with a variant of Sindbis virus (SIN) indicates that the lyticreplication of alphaviruses can be altered to suit the needs of the genetherapy application (Dryga, S. A. et al. (1997) Virology 228:74-83). Thewide host range of alphaviruses will allow the introduction of LIPAMinto a variety of cell types. The specific transduction of a subset ofcells in a population may require the sorting of cells prior totransduction. The methods of manipulating infectious cDNA clones ofalphaviruses, performing alphavirus cDNA and RNA transfections, andperforming alphavirus infections, are well known to those with ordinaryskill in the art.

[0256] Oligonucleotides derived from the transcription initiation site,e.g., between about positions −10 and +10 from the start site, may alsobe employed to inhibit gene expression. Similarly, inhibition can beachieved using triple helix base-pairing methodology. Triple helixpairing is useful because it causes inhibition of the ability of thedouble helix to open sufficiently for the binding of polymerases,transcription factors, or regulatory molecules. Recent therapeuticadvances using triplex DNA have been described in the literature. (See,e.g., Gee, J. E. et al. (1994) in Huber, B. E. and B. I. Carr, Molecularand Immunologic Approaches, Futura Publishing, Mt. Kisco N.Y., pp.163-177.) A complementary sequence or antisense molecule may also bedesigned to block translation of mRNA by preventing the transcript frombinding to ribosomes.

[0257] Ribozymes, enzymatic RNA molecules, may also be used to catalyzethe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules may specificallyand efficiently catalyze endonucleolytic cleavage of sequences encodingLIPAM.

[0258] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides, corresponding to the region of the target genecontaining the cleavage site, may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0259] Complementary ribonucleic acid molecules and ribozymes of theinvention may be prepared by any method known in the art for thesynthesis of nucleic acid molecules. These include techniques forchemically synthesizing oligonucleotides such as solid phasephosphoramidite chemical synthesis. Alternatively, RNA molecules may begenerated by in vitro and in vivo transcription of DNA sequencesencoding LIPAM. Such DNA sequences may be incorporated into a widevariety of vectors with suitable RNA polymerase promoters such as T7 orSP6. Alternatively, these cDNA constructs that synthesize complementaryRNA, constitutively or inducibly, can be introduced into cell lines,cells, or tissues.

[0260] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecule, or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

[0261] An additional embodiment of the invention encompasses a methodfor screening for a compound which is effective in altering expressionof a polynucleotide encoding LIPAM. Compounds which may be effective inaltering expression of a specific polynucleotide may include, but arenot limited to, oligonucleotides, antisense oligonucleotides, triplehelix-forming oligonucleotides, transcription factors and otherpolypeptide transcriptional regulators, and non-macromolecular chemicalentities which are capable of interacting with specific polynucleotidesequences. Effective compounds may alter polynucleotide expression byacting as either inhibitors or promoters of polynucleotide expression.Thus, in the treatment of disorders associated with increased LIPAMexpression or activity, a compound which specifically inhibitsexpression of the polynucleotide encoding LIPAM may be therapeuticallyuseful, and in the treatment of disorders associated with decreasedLIPAM expression or activity, a compound which specifically promotesexpression of the polynucleotide encoding LIPAM may be therapeuticallyuseful.

[0262] At least one, and up to a plurality, of test compounds may bescreened for effectiveness in altering expression of a specificpolynucleotide. A test compound may be obtained by any method commonlyknown in the art, including chemical modification of a compound known tobe effective in altering polynucleotide expression; selection from anexisting, commercially-available or proprietary library ofnaturally-occurring or non-natural chemical compounds; rational designof a compound based on chemical and/or structural properties of thetarget polynucleotide; and selection from a library of chemicalcompounds created combinatorially or randomly. A sample comprising apolynucleotide encoding LIPAM is exposed to at least one test compoundthus obtained. The sample may comprise, for example, an intact orpermeabilized cell, or an in vitro cell-free or reconstitutedbiochemical system. Alterations in the expression of a polynucleotideencoding LIPAM are assayed by any method commonly known in the art.Typically, the expression of a specific nucleotide is detected byhybridization with a probe having a nucleotide sequence complementary tothe sequence of the polynucleotide encoding LIPAM. The amount ofhybridization may be quantified, thus forming the basis for a comparisonof the expression of the polynucleotide both with and without exposureto one or more test compounds. Detection of a change in the expressionof a polynucleotide exposed to a test compound indicates that the testcompound is effective in altering the expression of the polynucleotide.A screen for a compound effective in altering expression of a specificpolynucleotide can be carried out, for example, using aSchizosaccharomyces pombe gene expression system (Atkins, D. et al.(1999) U.S. Pat. No. 5,932,435; Arndt, G. M. et al. (2000) Nucleic AcidsRes. 28:E15) or a human cell line such as HeLa cell (Clarke, M. L. etal. (2000) Biochem. Biophys. Res. Commun. 268:8-13). A particularembodiment of the present invention involves screening a combinatoriallibrary of oligonucleotides (such as deoxyribonucleotides,ribonucleotides, peptide nucleic acids, and modified oligonucleotides)for antisense activity against a specific polynucleotide sequence(Bruice, T. W. et al. (1997) U.S. Pat. No. 5,686,242; Bruice, T. W. etal. (2000) U.S. Pat. No. 6,022,691).

[0263] Many methods for introducing vectors into cells or tissues areavailable and equally suitable for use in vivo, in vitro, and ex vivo.For ex vivo therapy, vectors may be introduced into stem cells takenfrom the patient and clonally propagated for autologous transplant backinto that same patient. Delivery by transfection, by liposomeinjections, or by polycationic amino polymers may be achieved usingmethods which are well known in the art. (See, e.g., Goldman, C. K. etal. (1997) Nat. Biotechnol. 15:462-466.)

[0264] Any of the therapeutic methods described above may be applied toany subject in need of such therapy, including, for example, mammalssuch as humans, dogs, cats, cows, horses, rabbits, and monkeys.

[0265] An additional embodiment of the invention relates to theadministration of a composition which generally comprises an activeingredient formulated with a pharmaceutically acceptable excipient.Excipients may include, for example, sugars, starches, celluloses, gums,and proteins. Various formulations are commonly known and are thoroughlydiscussed in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing, Easton Pa.). Such compositions may consist of LIPAM,antibodies to LIPAM, and mimetics, agonists, antagonists, or inhibitorsof LIPAM.

[0266] The compositions utilized in this invention may be administeredby any number of routes including, but not limited to, oral,intravenous, intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal,intranasal, enteral, topical, sublingual, or rectal means.

[0267] Compositions for pulmonary administration may be prepared inliquid or dry powder form. These compositions are generally aerosolizedimmediately prior to inhalation by the patient. In the case of smallmolecules (e.g. traditional low molecular weight organic drugs), aerosoldelivery of fast-acting formulations is well-known in the art. In thcase of macromolecules (e.g. larger peptides and proteins), recentdevelopments in the field of pulmonary delivery via the alveolar regionof the lung have enabled the practical delivery of drugs such as insulinto blood circulation (see, e.g., Patton, J. S. et al., U.S. Pat. No.5,997,848). Pulmonary delivery has the advantage of administrationwithout needle injection, and obviates the need for potentially toxicpenetration enhancers.

[0268] Compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

[0269] Specialized forms of compositions may be prepared for directintracellular delivery of macromolecules comprising LIPAM or fragmentsthereof. For example, liposome preparations containing acell-impermeable macromolecule may promote cell fusion and intracellulardelivery of the macromolecule. Alternatively, LIPAM or a fragmentthereof may be joined to a short cationic N-terminal portion from theHIV Tat-1 protein. Fusion proteins thus generated have been found totransduce into the cells of all tissues, including the brain, in a mousemodel system (Schwarze, S. R. et al. (1999) Science 285:1569-1572).

[0270] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of neoplasticcells, or in animal models such as mice, rats, rabbits, dogs, monkeys,or pigs. An animal model may also be used to determine the appropriateconcentration range and route of administration. Such information canthen be used to determine useful doses and routes for administration inhumans.

[0271] A therapeutically effective dose refers to that amount of activeingredient, for example LIPAM or fragments thereof, antibodies of LIPAM,and agonists, antagonists or inhibitors of LIPAM, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orwith experimental animals, such as by calculating the ED₅₀ (the dosetherapeutically effective in 50% of the population) or LD₅₀ (the doselethal to 50% of the population) statistics. The dose ratio of toxic totherapeutic effects is the therapeutic index, which can be expressed asthe LD₅₀/ED₅₀ ratio. Compositions which exhibit large therapeuticindices are preferred. The data obtained from cell culture assays andanimal studies are used to formulate a range of dosage for human use.The dosage contained in such compositions is preferably within arange-of circulating concentrations that includes the ED₅₀ with littleor no toxicity. The dosage varies within this range depending upon thedosage form employed, the sensitivity of the patient, and the route ofadministration.

[0272] The exact dosag will be determined by the practitioner, in lightof factors related to the subject requiring treatment. Dosage andadministration ar adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, timeand frequency of administration, drug combination(s), reactionsensitivities, and response to therapy. Long-acting compositions may beadministered every 3 to 4 days, every week, or biweekly depending on thehalf-life and clearance rate of the particular formulation.

[0273] Normal dosage amounts may vary from about 0.1 μg to 100,000 μg,up to a total dose of about 1 gram, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

[0274] Diagnostics

[0275] In another embodiment, antibodies which specifically bind LIPAMmay be used for the diagnosis of disorders characterized by expressionof LIPAM, or in assays to monitor patients being treated with LIPAM oragonists, antagonists, or inhibitors of LIPAM. Antibodies useful fordiagnostic purposes may be prepared in the same manner as describedabove for therapeutics. Diagnostic assays for LIPAM include methodswhich utilize the antibody and a label to detect LIPAM in human bodyfluids or in extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by covalent ornon-covalent attachment of a reporter molecule. A wide variety ofreporter molecules, several of which are described above, are known inthe art and may be used.

[0276] A variety of protocols for measuring LIPAM, including ELISAs,RIAs, and FACS, are known in the art and provide a basis for diagnosingaltered or abnormal levels of LIPAM expression. Normal or standardvalues for LIPAM expression are established by combining body fluids orcell extracts taken from normal mammalian subjects, for example, humansubjects, with antibodies to LIPAM under conditions suitable for complexformation. The amount of standard complex formation may be quantitatedby various methods, such as photometric means. Quantities of LIPAMexpressed in subject, control, and disease samples from biopsied tissuesare compared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

[0277] In another embodiment of the invention, the polynucleotidesencoding LIPAM may be used for diagnostic purposes. The polynucleotideswhich may be used include oligonucleotide sequences, complementary RNAand DNA molecules, and PNAs. The polynucleotides may be used to detectand quantify gene expression in biopsied tissues in which expression ofLIPAM may be correlated with disease. The diagnostic assay may be usedto determine absence, presence, and excess expression of LIPAM, and tomonitor regulation of LIPAM levels during therapeutic intervention.

[0278] In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding LIPAM or closely related molecules may be used to identifynucleic acid sequences which encode LIPAM. The specificity of the probe,whether it is made from a highly specific region, e.g., the 5′regulatory region, or from a less specific region, e.g., a conservedmotif, and the stringency of the hybridization or amplification willdetermine whether the probe identifies only naturally occurringsequences encoding LIPAM, allelic variants, or related sequences.

[0279] Probes may also be used for the detection of related sequences,and may have at least 50% sequence identity to any of the LIPAM encodingsequences. The hybridization probes of the subject invention may be DNAor RNA and may be derived from the sequence of SEQ ID NO:10-18 or fromgenomic sequences including promoters, enhancers, and introns of theLIPAM gene.

[0280] Means for producing specific hybridization probes for DNAsencoding LIPAM include the cloning of polynucleotide sequences encodingLIPAM or LIPAM derivatives into vectors for the production of mRNAprobes. Such vectors are known in the art, are commercially available,and may be used to synthesize RNA probes in vitro by means of theaddition of the appropriate RNA polymerases and the appropriate labelednucleotides. Hybridization probes may be labeled by a variety ofreporter groups, for example, by radionuclides such as ³²P or ³⁵S, or byenzymatic labels, such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems, and the like.

[0281] Polynucleotide sequences encoding LIPAM may be used for thediagnosis of disorders associated with expression of LIPAM. Examples ofsuch disorders include, but are not limited to, a cancer, such asadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; a cardiovascular disordersuch as arteriovenous fistula, atherosclerosis, hypertension,vasculitis, Raynaud's disease, aneurysms, arterial dissections, varicoseveins, thrombophlebitis and phlebothrombosis, vascular tumors, andcomplications of thrombolysis, balloon angioplasty, vascularreplacement, and coronary artery bypass graft surgery, congestive heartfailure, ischemic heart disease, angina pectoris, myocardial infarction,hypertensive heart disease, degenerative valvular heart disease,calcific aortic valve stenosis, congenitally bicuspid aortic valve,mitral annular calcification, mitral valve prolapse, rheumatic fever andrheumatic heart disease, infective endocarditis, nonbacterial thromboticendocarditis, endocarditis of systemic lupus erythematosus, carcinoidheart disease, cardiomyopathy, myocarditis, pericarditis, neoplasticheart disease, congenital heart disease, and complications of cardiactransplantation, cong nital lung anomalies, atelectasis, pulmonarycongestion and edema, pulmonary embolism, pulmonary hemorrhage,pulmonary infarction, pulmonary hypertension, vascular sclerosis,obstructive pulmonary disease, restrictive pulmonary disease, chronicobstructive pulmonary disease, emphysema, chronic bronchitis, bronchialasthma, bronchiectasis, bacterial pneumonia, viral and mycoplasmalpneumonia, lung abscess, pulmonary tuberculosis, diffuse interstitialdiseases, pneumoconioses, sarcoidosis, idiopathic pulmonary fibrosis,desquamative interstitial pneumonitis, hypersensitivity pneumonitis,pulmonary eosinophilia bronchiolitis obliterans-organizing pneumonia,diffuse pulmonary hemorrhage syndromes, Goodpasture's syndromes,idiopathic pulmonary hemosiderosis, pulmonary involvement incollagen-vascular disorders, pulmonary alveolar proteinosis, lungtumors, inflammatory and noninflammatory pleural effusions,pneumothorax, pleural tumors, drug-induced lung disease,radiation-induced lung disease, and complications of lungtransplantation; a neurological disorder such as epilepsy, ischemiccerebrovascular disease, stroke, cerebral neoplasms, Alzheimer'sdisease, Pick's disease, Huntington's disease, dementia, Parkinson'sdisease and other extrapyramidal disorders, amyotrophic lateralsclerosis and other motor neuron disorders, progressive neural muscularatrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosisand other demyelinating diseases, bacterial and viral meningitis, brainabscess, subdural empyema, epidural abscess, suppurative intracranialthrombophlebitis, myelitis and radiculitis, viral central nervous systemdisease, prion diseases including kuru, Creutzfeldt-Jakob disease, andGerstmann-Straussler-Scheinker syndrome, fatal familial insomnia,nutritional and metabolic diseases of the nervous system,neurofibromatosis, tuberous sclerosis, cerebelloretinalhemangioblastomatosis, encephalotrigeminal syndrome, mental retardationand other developmental disorders of the central nervous systemincluding Down syndrome, cerebral palsy, neuroskeletal disorders,autonomic nervous system disorders, cranial nerve disorders, spinal corddiseases, muscular dystrophy and other neuromuscular disorders,peripheral nervous system disorders, dermatomyositis and polymyositis,inherited, metabolic, endocrine, and toxic myopathies, myastheniagravis, periodic paralysis, mental disorders including mood, anxiety,and schizophrenic disorders, seasonal affective disorder (SAD),akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia,dystonias, paranoid psychoses, postherpetic neuralgia, Tourette'sdisorder, progressive supranuclear palsy, corticobasal degeneration, andfamilial frontotemporal dementia; an autoimmune/inflammatory disordersuch as acquired immunodeficiency syndrome (AIDS), Addison's disease,adult respiratory distress syndrome, allergies, ankylosing spondylitis,amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolyticanemia, autoimmune thyroiditis, autoimmunepolyendocrinopathy-candidiasis-ectodermal dystrophy (APECED),bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopicdermatitis, dermatomyositis, diabetes mellitus, emphysema, episodiclymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythemanodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome,gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,irritable bowel syndrome, multiple sclerosis, myasthenia gravis,myocardial or pericardial inflammation, osteoarthritis, osteoporosis,pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoidarthritis, scleroderma, Sjbgren's syndrome, systemic anaphylaxis,systemic lupus erythematosus, systemic sclerosis, thrombocytopenicpurpura, ulcerative colitis, uveitis, Werner syndrome, complications ofcancer, hemodialysis, and extracorporeal circulation, viral, bacterial,fungal, parasitic, protozoal, and helminthic infections, and trauma; agastrointestinal disorder such as dysphagia, peptic esophagitis,esophageal spasm, esophageal stricture, esophageal carcinoma, dyspepsia,indigestion, gastritis, gastric carcinoma, anorexia, nausea, emesis,gastroparesis, antral or pyloric edema, abdominal angina, pyrosis,gastroenteritis, intestinal obstruction, infections of the intestinaltract, peptic ulcer, cholelithiasis, cholecystitis, cholestasis,pancreatitis, pancreatic carcinoma, biliary tract disease, hepatitis,hyperbilirubinemia, cirrhosis, passive congestion of the liver,hepatoma, infectious colitis, ulcerative colitis, ulcerative proctitis,Crohn's disease, Whipple's disease, Mallory-Weiss syndrome, coloniccarcinoma, colonic obstruction, irritable bowel syndrome, short bowelsyndrome, diarrhea, constipation, gastrointestinal hemorrhage, acquiredimmunodeficiency syndrome (AIDS) enteropathy, jaundice, hepaticencephalopathy, hepatorenal syndrome, hepatic steatosis,hemochromatosis, Wilson's disease, alpha,-antitrypsin deficiency, Reye'ssyndrome, primary sclerosing cholangitis, liver infarction, portal veinobstruction and thrombosis, centrilobular necrosis, peliosis hepatis,hepatic vein thrombosis, veno-occlusive disease, preeclampsia,eclampsia, acute fatty liver of pregnancy, intrahepatic cholestasis ofpregnancy, and hepatic tumors including nodular hyperplasias, adenomas,and carcinomas; and a disorder of lipid metabolism such as fatty liver,cholestasis, primary biliary cirrhosis, carnitine deficiency, carnitinepalmitoyltransferase deficiency, myoadenylate deaminase deficiency,hypertriglyceridemia, lipid storage disorders such Fabry's disease,Gaucher's disease, Niemann-Pick's disease, metachromatic leukodystrophy,adrenoleukodystrophy, GM₂ gangliosidosis, and ceroid lipofuscinosis,abetalipoproteinemia, Tangier disease, hyperlipoproteinemia, diabetesmellitus, lipodystrophy, lipomatoses, acute panniculitis, disseminatedfat necrosis, adiposis dolorosa, lipoid adrenal hyperplasia, minimalchange disease, lipomas, atherosclerosis, hypercholesterolemia,hypercholesterolemia with hypertriglyceridemia, primaryhypoalphalipoproteinemia, hypothyroidism, renal disease, liver disease,lecithin:cholesterol acyltransferase deficiency, cerebrotendinousxanthomatosis, sitosterolemia, hypocholesterolemia, Tay-Sachs disease,Sandhoff's disease, hyperlipidemia, hyperlipemia, lipid myopathies, andobesity. The polynucleotide sequences encoding LIPAM may be used inSouthern or northern analysis, dot blot, or other membrane-basedtechnologies; in PCR technologies; in dipstick, pin, and multiformatELISA-like assays; and in microarrays utilizing fluids or tissues frompatients to detect altered LIPAM expression. Such qualitative orquantitative methods are well known in the art.

[0282] In a particular aspect, the nucleotide sequences encoding LIPAMmay be useful in assays that detect the presence of associateddisorders, particularly those mentioned above. The nucleotide sequencesencoding LIPAM may be labeled by standard methods and added to a fluidor tissue sample from a patient under conditions suitable for theformation of hybridization complexes. After a suitable incubationperiod, the sample is washed and the signal is quantified and comparedwith a standard value. If the amount of signal in the patient sample issignificantly altered in comparison to a control sample then thepresence of altered levels of nucleotide sequences encoding LIPAM in thesample indicates the presence of the associated disorder. Such assaysmay also be used to evaluate the efficacy of a particular therapeutictreatment regimen in animal studies, in clinical trials, or to monitorthe treatment of an individual patient.

[0283] In order to provide a basis for the diagnosis of a disorderassociated with expression of LIPAM, a normal or standard profile forexpression is established. This may be accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with a sequence, or a fragment thereof, encoding LIPAM, underconditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with values from an experiment in which a known amountof a substantially purified polynucleotide is used. Standard valuesobtained in this manner may be compared with values obtained fromsamples from patients who are symptomatic for a disorder. Deviation fromstandard values is used to establish the presence of a disorder.

[0284] Once the presence of a disorder is established and a treatmentprotocol is initiated, hybridization assays may be repeated on a regularbasis to determine if the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0285] With respect to cancer, the presence of an abnormal amount oftranscript (either under- or overexpressed) in biopsied tissue from anindividual may indicate a predisposition for the development of thedisease, or may provide a means for detecting the disease prior to theappearance of actual clinical symptoms. A more definitive diagnosis ofthis type may allow health professionals to employ preventative measuresor aggressive treatment earlier thereby preventing the development orfurther progression of the cancer.

[0286] Additional diagnostic uses for oligonucleotides designed from thesequences encoding LIPAM may involve the use of PCR. These oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding LIPAM, or a fragment of a polynucleotide complementary to thepolynucleotide encoding LIPAM, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection orquantification of closely related DNA or RNA sequences.

[0287] In a particular aspect, oligonucleotide primers derived from thepolynucleotide sequences encoding LIPAM may be used to detect singlenucleotide polymorphisms (SNPs). SNPs are substitutions, insertions anddeletions that are a frequent cause of inherited or acquired geneticdisease in humans. Methods of SNP detection include, but are not limitedto, single-stranded conformation polymorphism (SSCP) and fluorescentSSCP (fSSCP) methods. In SSCP, oligonucleotide primers derived from thepolynucleotide sequences encoding LIPAM are used to amplify DNA usingthe polymerase chain reaction (PCR). The DNA may be derived, forexample, from diseased or normal tissue, biopsy samples, bodily fluids,and the like. SNPs in the DNA cause differences in the secondary andtertiary structures of PCR products in single-stranded form, and thesedifferences are detectable using gel electrophoresis in non-denaturinggels. In fSCCP, the oligonucleotide primers are fluorescently labeled,which allows detection of the amplimers in high-throughput equipmentsuch as DNA sequencing machines. Additionally, sequence databaseanalysis methods, termed in silico SNP (isSNP), are capable ofidentifying polymorphisms by comparing the sequence of individualoverlapping DNA fragments which assemble into a common consensussequence. These computer-based methods filter out sequence variationsdue to laboratory preparation of DNA and sequencing errors usingstatistical models and automated analyses of DNA sequence chromatograms.In the alternative, SNPs may be detected and characterized by massspectrometry using, for example, the high throughput MASSARRAY system(Sequenom, Inc., San Diego Calif.).

[0288] SNPs may be used to study the genetic basis of human disease. Forexample, at least 16 common SNPs have been associated withnon-insulin-dependent diabetes mellitus. SNPs are also useful forexamining differences in disease outcomes in monogenic disorders, suchas cystic fibrosis, sickle cell anemia, or chronic granulomatousdisease. For example, variants in the mannose-binding lectin, MBL2, havebeen shown to be correlated with deleterious pulmonary outcomes incystic fibrosis. SNPs also have utility in pharmacogenomics, theidentification of genetic variants that influence a patient's responseto a drug, such as life-threatening toxicity. For example, a variationin N-acetyl transferase is associated with a high incidence ofperipheral neuropathy in response to the anti-tuberculosis drugisoniazid, while a variation in the core promoter of the ALOX5 generesults in diminished clinical response to treatment with an anti-asthmadrug that targets the 5-lipoxygenase pathway. Analysis of thedistribution of SNPs in different populations is useful forinvestigating genetic drift, mutation, recombination, and selection, aswell as for tracing the origins of populations and their migrations.(Taylor, J. G. et al. (2001) Trends Mol. Med. 7:507-512; Kwok, P.-Y. andZ. Gu (1999) Mol. Med. Today 5:538-543; Nowotny, P. et al. (2001) Curr.Opin. Neurobiol. 11:637-641.)

[0289] Methods which may also be used to quantify the expression ofLIPAM include radiolabeling or biotinylating nucleotides,coamplification of a control nucleic acid, and interpolating resultsfrom standard curves. (See, e.g., Melby, P. C. et al. (1993) J. Immunol.Methods 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem.212:229-236.) The speed of quantitation of multiple samples may beaccelerated by running the assay in a high-throughput format where theoligomer or polynucleotide of interest is presented in various dilutionsand a spectrophotometric or colorimetric response gives rapidquantitation.

[0290] In further embodiments, oligonucleotides or longer fragmentsderived from any of the polynucleotide sequences described herein may beused as elements on a microarray. The microarray can be used intranscript imaging techniques which monitor the relative expressionlevels of large numbers of genes simultaneously as described below. Themicroarray may also be used to identify genetic variants, mutations, andpolymorphisms. This information may be used to determine gene function,to understand the genetic basis of a disorder, to diagnose a disorder,to monitor progression/regression of disease as a function ofgene-expression, and to develop and monitor the activities oftherapeutic agents in the treatment of disease. In particular, thisinformation may be used to develop a pharmacogenomic profile of apatient in order to select the most appropriate and effective treatmentregimen for that patient. For example, therapeutic agents which arehighly effective and display the fewest side effects may be selected fora patient based on his/her pharmacogenomic profile.

[0291] In another embodiment, LIPAM, fragments of LIPAM, or antibodiesspecific for LIPAM may be used as elements on a microarray. Themicroarray may be used to monitor or measure protein-proteininteractions, drug-target interactions, and gene expression profiles, asdescribed above.

[0292] A particular embodiment relates to the use of the polynucleotidesof the present invention to generate a transcript image of a tissue orcell type. A transcript image represents the global pattern of geneexpression by a particular tissue or cell type. Global gene expressionpatterns are analyzed by quantifying the number of expressed genes andtheir relative abundance under given conditions and at a given time.(See Seilhamer et al., “Comparative Gene Transcript Analysis,” U.S. Pat.No. 5,840,484, expressly incorporated by reference herein.) Thus atranscript image may be generated by hybridizing the polynucleotides ofthe present invention or their complements to the totality oftranscripts or reverse transcripts of a particular tissue or cell type.In one embodiment, the hybridization takes place in high-throughputformat, wherein the polynucleotides of the present invention or theircomplements comprise a subset of a plurality of elements on amicroarray. The resultant transcript image would provide a profile ofgene activity.

[0293] Transcript images may be generated using transcripts isolatedfrom tissues, cell lines, biopsies, or other biological samples. Thetranscript image may thus reflect gene expression in vivo, as in thecase of a tissue or biopsy sample, or in vitro, as in the case of a cellline.

[0294] Transcript images which profile the expression of thepolynucleotides of the present invention may also be used in conjunctionwith in vitro model systems and preclinical evaluation ofpharmaceuticals, as well as toxicological testing of industrial andnaturally-occurring environmental. compounds. All compounds inducecharacteristic gene expression patterns, frequently termed molecularfingerprints or toxicant signatures, which are indicative of mechanismsof action and toxicity (Nuwaysir, E. F. et al. (1999) Mol. Carcinog.24:153-159; Steiner, S. and N. L. Anderson (2000) Toxicol. Lett.112-113:467-471, expressly incorporated by reference herein). If a testcompound has a signature similar to that of a compound with knowntoxicity, it is likely to share those toxic properties. Thesefingerprints or signatures are most useful and refined when they containexpression information from a large number of genes and gene families.Ideally, a genome-wide measurement of expression provides the highestquality signature. Even genes whose expression is not altered by anytested compounds are important as well, as the levels of expression ofthese genes are used to normalize the rest of the expression data. Thenormalization procedure is useful for comparison of expression dataafter treatment with different compounds. While the assignment of genefunction to elements of a toxicant signature aids in interpretation oftoxicity mechanisms, knowledge of gene function is not necessary for thestatistical matching of signatures which leads to prediction oftoxicity. (See, for example, Press Release 00-02 from the NationalInstitute of Environmental Health Sciences, released Feb. 29, 2000,available at http://www.niehs.nih.gov/oc/news/toxchip.htm.) Therefore,it is important and desirable in toxicological screening using toxicantsignatures to include all expressed gene sequences.

[0295] In one embodiment, the toxicity of a test compound is assessed bytreating a biological sample containing nucleic acids with the testcompound. Nucleic acids that are expressed in the treated biologicalsample are hybridized with one or more probes specific to thepolynucleotides of the present invention, so that transcript levelscorresponding to the polynucleotides of the present invention may bequantified. The transcript levels in the treated biological sample arecompared with levels in an untreated biological sample. Differences inthe transcript levels between the two samples are indicative of a toxicresponse caused by the test compound in the treated sample.

[0296] Another particular embodiment relates to the use of thepolypeptide sequences of the present invention to analyze the proteomeof a tissue or cell type. The term proteome refers to the global patternof protein expression in a particular tissue or cell type. Each proteincomponent of a proteome can be subjected individually to furtheranalysis. Proteome expression patterns, or profiles, are analyzed byquantifying the number of expressed proteins and their relativeabundance under given conditions and at a given time. A profile of acell's proteome may thus be generated by separating and analyzing thepolypeptides of a particular tissue or cell type. In one embodiment, theseparation is achieved using two-dimensional gel electrophoresis, inwhich proteins from a sample are separated by isoelectric focusing inthe first dimension, and then according to molecular weight by sodiumdodecyl sulfate slab gel electrophoresis in the second dimension(Steiner and Anderson, supra). The proteins are visualized in the gel asdiscrete and uniquely positioned spots, typically by staining the gelwith an agent such as Coomassie Blue or silver or fluorescent stains.The optical density of each protein spot is generally proportional tothe level of the protein in the sample. The optical densities ofequivalently positioned protein spots from different samples, forexample, from biological samples either treated or untreated with a testcompound or therapeutic agent, are compared to identify any changes inprotein spot density related to the treatment. The proteins in the spotsare partially sequenced using, for example, standard methods employingchemical or enzymatic cleavage followed by mass spectrometry. Theidentity of the protein in a spot may be determined by comparing itspartial sequence, preferably of at least 5 contiguous amino acidresidues, to the polypeptide sequences of the present invention. In somecases, further sequence data may be obtained for definitive proteinidentification.

[0297] A proteomic profile may also be generated using antibodiesspecific for LIPAM to quantify the levels of LIPAM expression. In oneembodiment, the antibodies are used as elements on a microarray, andprotein expression levels are quantified by exposing the microarray tothe sample and detecting the levels of protein bound to each arrayelement (Lueking, A. et al. (1999) Anal. Biochem. 270:103-111; Mendoze,L. G. et al. (1999) Biotechniques 27:778-788). Detection may beperformed by a variety of methods known in the art, for example, byreacting the proteins in the sample with a thiol- or amino-reactivefluorescent compound and detecting the amount of fluorescence bound ateach array element.

[0298] Toxicant signatures at the proteome level are also useful fortoxicological screening, and should be analyzed in parallel withtoxicant signatures at the transcript level. There is a poor correlationbetween transcript and protein abundances for some proteins in sometissues (Anderson, N. L. and J. Seilhamer (1997) Electrophoresis18:533-537), so proteome toxicant signatures may be useful in theanalysis of compounds which do not significantly affect the transcriptimage, but which alter the proteomic profile. In addition, the analysisof transcripts in body fluids is difficult, due to rapid degradation ofmRNA, so proteomic profiling may be more reliable and informative insuch cases.

[0299] In another embodiment, the toxicity of a test compound isassessed by treating a biological sample containing proteins with thetest compound. Proteins that are expressed in the treated biologicalsample are separated so that the amount of each protein can bequantified. The amount of each protein is compared to the amount of thecorresponding protein in an untreated biological sample. A difference inthe amount of protein between the two samples is indicative of a toxicresponse to the test compound in the treated sample. Individual proteinsare identified by sequencing the amino acid residues of the individualproteins and comparing these partial sequences to the polypeptides ofthe present invention.

[0300] In another embodiment, the toxicity of a test compound isassessed by treating a biological sample containing proteins with thetest compound. Proteins from the biological sample are incubated withantibodies specific to the polypeptides of the present invention. Theamount of protein recognized by the antibodies is quantified. The amountof protein in the treated biological sample is compared with the amountin an untreated biological sample. A difference in the amount of proteinbetween the two samples is indicative of a toxic response to the testcompound in the treated. sample.

[0301] Microarrays may be prepared, used, and analyzed using methodsknown in the art. (See, e.g., Brennan, T. M. et al. (1995) U.S. Pat. No.5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. USA93:10614-10619; Baldeschweiler et al. (1995) PCT applicationWO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505;Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2150-2155; andHeller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.) Various types ofmicroarrays are well known and thoroughly described in DNA Microarrays:A Practical Approach, M. Schena, ed. (1999) Oxford University Press,London, hereby expressly incorporated by reference.

[0302] In another embodiment of the invention, nucleic acid sequencesencoding LIPAM may be used to generate hybridization probes useful inmapping the naturally occurring genomic sequence. Either coding ornoncoding sequences may be used, and in some instances, noncodingsequences may be preferable over coding sequences. For example,conservation of a coding sequence among members of a multi-gene familymay potentially cause undesired cross hybridization during chromosomalmapping. The sequences may be mapped to a particular chromosome, to aspecific region of a chromosome, or to artificial chromosomeconstructions, e.g., human artificial chromosomes (HACs), yeastartificial chromosomes (YACs), bacterial artificial chromosomes (BACs),bacterial P1 constructions, or single chromosome cDNA libraries. (See,e.g., Harrington, J. J. et al. (1997) Nat. Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B. J. (1991) Trends Genet.7:149-154.) Once mapped, the nucleic acid sequences of the invention maybe used to develop genetic linkage maps, for example, which correlatethe inheritance of a disease state with the inheritance of a particularchromosome region or restriction fragment length polymorphism (RFLP).(See, for example, Lander, E. S. and D. Botstein (1986) Proc. Natl.Acad. Sci. USA 83:7353-7357.)

[0303] Fluorescent in situ hybridization (FISH) may be correlated withother physical and genetic map data. (See, e.g., Heinz-Ulrich, et al.(1995) in Meyers, supra, pp. 965-968.) Examples of genetic map data canbe found in various scientific journals or at the Online MendelianInheritance in Man (OMIM) World Wide Web site. Correlation between thelocation of the gene encoding LIPAM on a physical map and a specificdisorder, or a predisposition to a specific disorder, may help definethe region of DNA associated with that disorder and thus may furtherpositional cloning efforts.

[0304] In situ hybridization of chromosomal preparations and physicalmapping techniques, such as linkage analysis using establishedchromosomal markers, may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the exact chromosomallocus is not known. This information is valuable to investigatorssearching for disease genes using positional cloning or other genediscovery techniques. Once the gene or genes responsible for a diseaseor syndrome have been crudely localized by genetic linkage to aparticular genomic region, e.g., ataxia-telangiectasia to 11q22-23, anysequences mapping to that area may represent associated or regulatorygenes for further investigation. (See, e.g., Gatti, R. A. et al. (1988)Nature 336:577-580.) The nucleotide sequence of the instant inventionmay also be used to detect differences in the chromosomal location dueto translocation, inversion, etc., among normal, carrier, or affectedindividuals.

[0305] In another embodiment of the invention, LIPAM, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes betweenLIPAM and the agent being tested may be m asured.

[0306] Another technique for drug screening provides for high throughputscreening of compounds having suitable binding affinity to the proteinof interest. (See, e.g., Geysen, et al. (1984) PCT applicationWO84/03564.) In this method, large numbers of different small testcompounds are synthesized on a solid substrate. The test compounds arereacted with LIPAM, or fragments thereof, and washed. Bound LIPAM isthen detected by methods well known in the art. Purified LIPAM can alsobe coated directly onto plates for use in the aforementioned drugscreening techniques. Alternatively, non-neutralizing antibodies can beused to capture the peptide and immobilize it on a solid support.

[0307] In another embodiment, one may use competitive drug screeningassays in which neutralizing antibodies capable of binding LIPAMspecifically compete with a test compound for binding LIPAM. In thismanner, antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with LIPAM.

[0308] In additional embodiments, the nucleotide sequences which encodeLIPAM may be used in any molecular biology techniques that have yet tobe developed, provided the new techniques rely on properties ofnucleotide sequences that are currently known, including, but notlimited to, such properties as the triplet genetic code and specificbase pair interactions.

[0309] Without further elaboration, it is believed that one skilled inthe art can, using the preceding description, utilize the presentinvention to its fullest extent. The following embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe remainder of the disclosure in any way whatsoever.

[0310] The disclosures of all patents, applications and publications,mentioned above and below, including U.S. Ser. No. 60/266,910, U.S. Ser.No. 60/276,891, U.S. Ser. No. 60/279,760, U.S. Ser. No. 60/283,818, U.S.Ser. No. 60/276,855, and U.S. Ser. No. 60/285,405, are expresslyincorporated by reference herein.

EXAMPLES

[0311] I. Construction of cDNA Libraries

[0312] Incyte cDNAs were derived from cDNA libraries described in theLIFESEQ GOLD database (Incyte Genomics, Palo Alto Calif.). Some tissueswere homogenized and lysed in guanidinium isothiocyanate, while otherswere homogenized and lysed in phenol or in a suitable mixture ofdenaturants, such as TRIZOL (Life Technologies), a monophasic solutionof phenol and guanidine isothiocyanate. The resulting lysates werecentrifuged over CsCl cushions or extracted with chloroform. RNA wasprecipitated from the lysates with either isopropanol or sodium acetateand ethanol, or by other routine methods.

[0313] Phenol extraction and precipitation of RNA were repeated asnecessary to increase RNA purity. In some cases, RNA was treated withDNase. For most libraries, poly(A)+RNA was isolated using oligod(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles(QIAGEN, Chatsworth Calif.), or an OLIGOTEX mRNA purification kit(QIAGEN). Alternatively, RNA was isolated directly from tissue lysatesusing other RNA isolation kits, e.g., the POLY(A)PURE mRNA purificationkit (Ambion, Austin Tex.).

[0314] In some cases, Stratagene was provided with RNA and constructedthe corresponding cDNA libraries. Otherwise, cDNA was synthesized andcDNA libraries were constructed with the UNIZAP vector system(Stratagene) or SUPERSCRIPT plasmid system (Life Technologies), usingthe recommended procedures or similar methods known in the art. (See,e.g., Ausubel, 1997, supra, units 5.1-6.6.) Reverse transcription wasinitiated using oligo d(T) or random primers. Synthetic oligonucleotideadapters were ligated to double stranded cDNA, and the cDNA was digestedwith the appropriate restriction enzyme or enzymes. For most libraries,the cDNA was size-selected (300-1000 bp) using SEPHACRYL S1000,SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (AmershamPharmacia Biotech) or preparative agarose gel electrophoresis. cDNAswere ligated into compatible restriction enzyme sites of the polylinkerof a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1plasmid (Life Technologies), PCDNA2.1 plasmid (Invitrogen, CarlsbadCalif.), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen),PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo AltoCalif.), pRARE (Incyte Genomics), or pINCY (Incyte Genomics), orderivatives thereof. Recombinant plasmids were transformed intocompetent E. coli cells including XL1-Blue, XL1-BlueMRF, or SOLR fromStratagene or DH5α, DH10B, or ElectroMAX DH10B from Life Technologies.

[0315] II. Isolation of cDNA Clones

[0316] Plasmids obtained as described in Example I were recovered fromhost cells by in vivo excision using the UNIZAP vector system(Stratagene) or by cell lysis. Plasmids were purified using at least oneof the following: a Magic or WIZARD Minipreps DNA purification system(Promega); an AGTC Miniprep purification kit (Edge Biosystems,Gaithersburg Md.); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid,QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP 96plasmid purification kit from QIAGEN. Following precipitation, plasmidswere resuspended in 0.1 ml of distilled water and stored, with orwithout lyophilization, at 4° C.

[0317] Alternatively, plasmid DNA was amplified from host cell lysatesusing direct link PCR in a high-throughput format (Rao, V. B. (1994)Anal. Biochem. 216:1-14). Host cell lysis and thermal cycling steps werecarried out in a single reaction mixture. Samples were processed andstored in 384-well plates, and the concentration of amplified plasmidDNA was quantified fluorometrically using PICOGREEN dye (MolecularProbes, Eugene Oreg.) and a FLUOROSKAN II fluorescence scanner(Labsystems Oy, Helsinki, Finland).

[0318] III. Sequencing and Analysis

[0319] Incyte cDNA recovered in plasmids as described in Example II weresequenced as follows. Sequencing reactions were processed using standardmethods or high-throughput instrumentation such as the ABI CATALYST 800(Applied Biosystems) thermal cycler or the PTC-200 thermal cycler (MJResearch) in conjunction with the HYDRA microdispenser (RobbinsScientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNAsequencing reactions were prepared using reagents provided by AmershamPharmacia Biotech or supplied in ABI sequencing kits such as the ABIPRISM BIGDYE Terminator cycle sequencing ready reaction kit (AppliedBiosystems). Electrophoretic separation of cDNA sequencing reactions anddetection of labeled polynucleotides were carried out using the MEGABACE1000 DNA sequencing system (Molecular Dynamics); the ABI PRISM 373 or377 sequencing system (Applied Biosystems) in conjunction with standardABI protocols and base calling software; or other sequence analysissystems known in the art. Reading frames within the cDNA sequences wereidentified using standard methods (reviewed in Ausubel, 1997, supra,unit 7.7). Some of the cDNA sequences were selected for extension usingthe techniques disclosed in Example VIII.

[0320] The polynucleotide sequences derived from Incyte cDNAs werevalidated by removing vector, linker, and poly(A) sequences and bymasking ambiguous bases, using algorithms and programs based on BLAST,dynamic programming, and dinucleotide nearest neighbor analysis. TheIncyte cDNA sequences or translations thereof were then queried againsta selection of public databases such as the GenBank primate, rodent,mammalian, vertebrate, and eukaryote databases, and BLOCKS, PRINTS,DOMO, PRODOM; PROTEOME databases with sequences from Homo sapiens,Rattus norvegicus, Mus musculus, Caenorhabditis elegans, Saccharomycescerevisiae, Schizosaccharomyces pombe, and Candida albicans (IncyteGenomics, Palo Alto Calif.); and hidden Markov model (HMM)-based proteinfamily databases such as PFAM. (HMM is a probabilistic approach whichanalyzes consensus primary structures of gene families. See, forexample, Eddy, S. R. (1996) Curr. Opin. Struct. Biol. 6:361-365.) Thequeries were performed using programs based on BLAST, FASTA, BLIMPS, andHMMER. The Incyte cDNA sequences were assembled to produce full lengthpolynucleotide sequences. Alternatively, GenBank cDNAs, GenBank ESTs,stitched sequences, stretched sequences, or Genscan-predicted codingsequences (see Examples IV and V) were used to extend Incyte cDNAassemblages to full length. Assembly was performed using programs basedon Phred, Phrap, and Consed, and cDNA assemblages were screened for openreading frames using programs based on GeneMark, BLAST, and FASTA. Thefull length polynucleotide sequences were translated to derive thecorresponding full length polypeptide sequences. Alternatively, apolypeptide of the invention may begin at any of the methionine residuesof the full length translated polypeptide. Full length polypeptidesequences were subsequently analyzed by querying against databases suchas the GenBank protein databases (genpept), SwissProt, the PROTEOMEdatabases, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, and hidden Markovmodel (HMM)-based protein family databases such as PFAM. Full lengthpolynucleotide sequences are also analyzed using MACDNASIS PRO software(Hitachi Software Engineering, South San Francisco CA) and LASERGENEsoftware (DNASTAR). Polynucleotide and polypeptide sequence alignmentsare generated using default parameters specified by the CLUSTALalgorithm as incorporated into the MEGALIGN multisequence alignmentprogram (DNASTAR), which also calculates the percent identity betweenaligned sequences.

[0321] Table 7 summarizes the tools, programs, and algorithms used forthe analysis and assembly of Incyte cDNA and full length sequences andprovides applicable descriptions, references, and threshold parameters.The first column of Table 7 shows the tools, programs, and algorithmsused, the second column provides brief descriptions thereof, the thirdcolumn presents appropriate references, all of which are incorporated byreference herein in their entirety, and the fourth column presents,where applicable, the scores, probability values, and other parametersused to evaluate the strength of a match between two sequences (thehigher the score or the lower the probability value, the greater theidentity between two sequences).

[0322] The programs described above for the assembly and analysis offull length polynucleotide and polypeptide sequences were also used toidentify polynucleotide sequence fragments from SEQ ID NO:10-18.Fragments from about 20 to about 4000 nucleotides which are useful inhybridization and amplification technologies are described in Table 4,column 2.

[0323] IV. Identification and Editing of Coding Sequences from GenomicDNA

[0324] Putative lipid-associated molecules were initially identified byrunning the Genscan gene identification program against public genomicsequence databases (e.g., gbpri and gbhtg). Genscan is a general-purposegene identification program which analyzes genomic DNA sequences from avariety of organisms (See Burge, C. and S. Karlin (1997) J. Mol. Biol.268:78-94, and Burge, C. and S. Karlin (1998) Curr. Opin. Struct. Biol.8:346-354). The program concatenates predicted exons to form anassembled cDNA sequence extending from a methionine to a stop codon. Theoutput of Genscan is a FASTA database of polynucleotide and polypeptidesequences. The maximum range of sequence for Genscan to analyze at oncewas set to 30 kb. To determine which of these Genscan predicted cDNAsequences encode lipid-associated molecules, the encoded polypeptideswere analyzed by querying against PFAM models for lipid-associatedmolecules. Potential lipid-associated molecules were also identified byhomology to Incyte cDNA sequences that had been annotated aslipid-associated molecules. These selected Genscan-predicted sequenceswere then compared by BLAST analysis to the genpept and gbpri publicdatabases. Where necessary, the Genscan-predicted sequences were thenedited by comparison to the top BLAST hit from genpept to correct errorsin the sequence predicted by Genscan, such as extra or omitted exons.BLAST analysis was also used to find any Incyte cDNA or public cDNAcoverage of the Genscan-predicted sequences, thus providing evidence fortranscription. When Incyte cDNA coverage was available, this informationwas used to correct or confirm the Genscan predicted sequence. Fulllength polynucleotide sequences were obtained by assemblingGenscan-predicted coding sequences with Incyte cDNA sequences and/orpublic cDNA sequences using the assembly process described in ExampleIII. Alternatively, full length polynucleotide sequences were derivedentirely from edited or unedited Genscan-predicted coding sequences.

[0325] V. Assembly of Genomic Sequence Data with cDNA Sequence Data

[0326] “Stitched” Sequences

[0327] Partial cDNA sequences were extended with exons predicted by theGenscan gene identification program described in Example IV. PartialcDNAs assembled as described in Example III were mapped to genomic DNAand parsed into clusters containing related cDNAs and Genscan exonpredictions from one or more genomic sequences. Each cluster wasanalyzed using an algorithm based on graph theory and dynamic programingto integrate cDNA and genomic information, generating possible splicevariants that were subsequently confirmed, edited, or extended to createa full length sequence. Sequence intervals in which the entire length ofthe interval was present on more than one sequence in the cluster wereidentified, and intervals thus identified were considered to beequivalent by transitivity. For example, if an interval was present on acDNA and two genomic sequences, then all three intervals were consideredto be equivalent. This process allows unrelated but consecutive genomicsequences to be brought together, bridged by cDNA sequence. Intervalsthus identified were then “stitched” together by the stitching algorithmin the order that they appear along their parent sequences to generatethe longest possible sequence, as well as sequence variants. Linkagesbetween intervals which proceed along one type of parent sequence (cDNAto cDNA or genomic sequence to genomic sequence) were given preferenceover linkages which change parent type (cDNA to genomic sequence). Theresultant stitched sequences were translated and compared by BLASTanalysis to the genpept and gbpri public databases. Incorrect exonspredicted by Genscan were corrected by comparison to the top BLAST hitfrom genpept. Sequences were further extended with additional cDNAsequences, or by inspection of genomic DNA, when necessary.

[0328] “Stretched” Sequences

[0329] Partial DNA sequences were extended to full length with analgorithm based on BLAST analysis. First, partial cDNAs assembled asdescribed in Example III were queried against public databases such asthe GenBank primate, rodent, mammalian, vertebrate, and eukaryotedatabases using the BLAST program. The nearest GenBank protein homologwas then compared by BLAST analysis to either Incyte cDNA sequences orGenScan exon predicted sequences described in Example IV. A chimericprotein was generated by using the resultant high-scoring segment pairs(HSPs) to map the translated sequences onto the GenBank protein homolog.Insertions or deletions may occur in the chimeric protein with respectto the original GenBank protein homolog. The GenBank protein homolog,the chimeric protein, or both were used as probes to search forhomologous genomic sequences from the public human genome databases.Partial DNA sequences were therefore “stretched” or extended by theaddition of homologous genomic sequences. The resultant stretchedsequences were examined to determine whether it contained a completegene.

[0330] VI. Chromosomal Mapping of LIPAM Encoding Polynucleotides

[0331] The sequences which were used to assemble SEQ ID NO:10-18 werecompared with sequences from the Incyte LIFESEQ database and publicdomain databases using BLAST and other implementations of theSmith-Waterman algorithm. Sequences from these databases that matchedSEQ ID NO:10-18 were assembled into clusters of contiguous andoverlapping sequences using assembly algorithms such as Phrap (Table 7).Radiation hybrid and genetic mapping data available from publicresources such as the Stanford Human Genome Center (SHGC), WhiteheadInstitute for Genome Research (WIGR), and Généthon were used todetermine if any of the clustered sequences had been previously mapped.Inclusion of a mapped sequence in a cluster resulted in the assignmentof all sequences of that cluster, including its particular SEQ ID NO:,to that map location.

[0332] Map locations are represented by ranges, or intervals, of humanchromosomes. The map position of an interval, in centiMorgans, ismeasured relative to the terminus of the chromosome's p-arm. (ThecentiMorgan (cM) is a unit of measurement based on recombinationfrequencies between chromosomal markers. On average, 1 cM is roughlyequivalent to 1 megabase (Mb) of DNA in humans, although this can varywidely due to hot and cold spots of recombination.) The cM distances arebased on genetic markers mapped by Généthon which provide boundaries forradiation hybrid markers whose sequences were included in each of theclusters. Human genome maps and other resources available to the public,such as the NCBI “GeneMap'99” World Wide Web site(http://www.ncbi.nlm.nih.gov/genemap/), can be employed to determine ifpreviously identified disease genes map within or in proximity to theintervals indicated above.

[0333] VII. Analysis of Polynucleotide Expression

[0334] Northern analysis is a laboratory technique used to detect thepresence of a transcript of a gene and involves the hybridization of alabeled nucleotide sequence to a membrane on which RNAs from aparticular cell type or tissue have been bound. (See, e.g., Sambrook,supra, ch. 7; Ausubel (1995) supra, ch. 4 and 16.)

[0335] Analogous computer techniques applying BLAST were used to searchfor identical or related molecules in cDNA databases such as GenBank orLIFESEQ (Incyte Genomics). This analysis is much faster than multiplemembrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or similar. The basis of the search is theproduct score, which is defined as:$\frac{{BLAST}\quad {Score} \times {Percent}\quad {Identity}}{5 \times {minimum}\quad \left\{ {{{length}\quad \left( {{Seq}.\quad 1} \right)},{{length}\quad \left( {{Seq}.\quad 2} \right)}} \right\}}$

[0336] The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.The product score is a normalized value between 0 and 100, and iscalculated as follows: the BLAST score is multiplied by the percentnucleotide identity and the product is divided by (5 times the length ofthe shorter of the two sequences). The BLAST score is calculated byassigning a score of +5 for every base that matches in a high-scoringsegment pair (HSP), and −4 for every mismatch. Two sequences may sharemore than one HSP (separated by gaps). If there is more than one HSP,then the pair with the highest BLAST score is used to calculate theproduct score. The product score represents a balance between fractionaloverlap and quality in a BLAST alignment. For example, a product scoreof 100 is produced only for 100% identity over the entire length of theshorter of the two sequences being compared. A product score of 70 isproduced either by 100% identity and 70% overlap at one end, or by 88%identity and 100% overlap at the other. A product score of 50 isproduced either by 100% identity and 50% overlap at one end, or 79%identity and 100% overlap.

[0337] Alternatively, polynucleotide sequences encoding LIPAM areanalyzed with respect to the tissue sources from which they werederived. For example, some full length sequences are assembled, at leastin part, with overlapping Incyte cDNA sequences (see Example III). EachcDNA sequence is derived from a cDNA library constructed from a humantissue. Each human tissue is classified into one of the followingorgan/tissue categories: cardiovascular system; connective tissue;digestive system; embryonic structures; endocrine system; exocrineglands; genitalia, female; genitalia, male; germ cells; hemic and immunesystem; liver; musculoskeletal system; nervous system; pancreas;respiratory system; sense organs; skin; stomatognathic system;unclassified/mixed; or urinary tract. The number of libraries in eachcategory is counted and divided by the total number of libraries acrossall categories. Similarly, each human tissue is classified into one ofthe following disease/condition categories: cancer, cell line,developmental, inflammation, neurological, trauma, cardiovascular,pooled, and other, and the number of libraries in each category iscounted and divided by the total number of libraries across allcategories. The resulting percentages reflect the tissue- anddisease-specific expression of cDNA encoding LIPAM. cDNA sequences andcDNA library/tissue information are found in the LIFESEQ GOLD database(Incyte Genomics, Palo Alto Calif.).

[0338] VIII. Extension of LIPAM Encoding Polynucleotides

[0339] Full length polynucleotide sequences were also produced byextension of an appropriate fragment of the full length molecule usingoligonucleotide primers designed from this fragment. One primer wassynthesized to initiate 5′ extension of the known fragment, and theother primer was synthesized to initiate 3′ extension of the knownfragment. The initial primers were designed using OLIGO 4.06 software(National Biosciences), or another appropriate program, to be about 22to 30 nucleotides in length, to have a GC content of about 50% or more,and to anneal to the target sequence at temperatures of about 68° C. toabout 72° C. Any stretch of nucleotides which would result in hairpinstructures and primer-primer dimerizations was avoided.

[0340] Selected human cDNA libraries were used to extend the sequence.If more than one extension was necessary or desired, additional ornested sets of primers were designed.

[0341] High fidelity amplification was obtained by PCR using methodswell known in the art. PCR was performed in 96-well plates using thePTC-200 thermal cycler (MJ Research, Inc.). The reaction mix containedDNA template, 200 nmol of each primer, reaction buffer containing Mg²⁺,(NH₄)₂SO₄, and 2-mercaptoethanol, Taq DNA polymerase (Amersham PharmaciaBiotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase(Stratagene), with the following parameters for primer pair PCI A andPCI B: Step 1: 94° C., 3 min; Step 2: 94° C., 15 sec; Step 3: 60° C., 1min; Step 4: 68° C., 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times;Step 6: 68° C., 5 min; Step 7: storage at 4° C. In the alternative, theparameters for primer pair T7 and SK+ were as follows: Step 1: 94° C., 3min; Step 2: 94° C., 15 sec; Step 3: 57° C., 1 min; Step 4: 68° C., 2min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68° C., 5 min;Step 7: storage at 4° C.

[0342] The concentration of DNA in each well was determined bydispensing 100 μl PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN;Molecular Probes, Eugene Oreg.) dissolved in 1×TE and 0.5 μl ofundiluted PCR product into each well of an opaque fluorimeter plate(Corning Costar, Acton Mass.), allowing the DNA to bind to the reagent.The plate was scanned in a Fluoroskan II (Labsystems Oy, Helsinki,Finland) to measure the fluorescence of the sample and to quantify theconcentration of DNA. A 5 μl to 10 μl aliquot of the reaction mixturewas analyzed by electrophoresis on a 1% agarose gel to determine whichreactions were successful in extending the sequence.

[0343] The extended nucleotides were desalted and concentrated,transferred to 384-well plates, digested with CviJI cholera virusendonuclease (Molecular Biology Research, Madison Wis.), and sonicatedor sheared prior to religation into pUC 18 vector (Amersham PharmaciaBiotech). For shotgun sequencing, the digested nucleotides wereseparated on low concentration (0.6 to 0.8%) agarose gels, fragmentswere excised, and agar digested with Agar ACE (Promega). Extended cloneswere religated using T4 ligase (New England Biolabs, Beverly Mass.) intopUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNApolymerase (Stratagene) to fill-in restriction site overhangs, andtransfected into competent E. coli cells. Transformed cells wereselected on antibiotic-containing media, and individual colonies werepicked and cultured overnight at 37° C. in 384-well plates in LB/2× carbliquid media.

[0344] The cells were lysed, and DNA was amplified by PCR using Taq DNApolymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase(Stratagene) with the following parameters: Step 1: 94° C., 3 min; Step2: 94° C., 15 sec; Step 3: 60° C., 1 min; Step 4: 72° C., 2 min; Step 5:steps 2, 3, and 4 repeated 29 times; Step 6: 72° C., 5 min; Step 7:storage at 4° C. DNA was quantified by PICOGREEN reagent (MolecularProbes) as described above. Samples with low DNA recoveries werereamplified using the same conditions as described above. Samples werediluted with 20% dimethysulfoxide (1:2, v/v), and sequenced usingDYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit(Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator cyclesequencing ready reaction kit (Applied Biosystems).

[0345] In like manner, full length polynucleotide sequences are verifiedusing the above procedure or are used to obtain 5′ regulatory sequencesusing the above procedure along with oligonucleotides designed for suchextension, and an appropriate genomic library.

[0346] IX. Identification of Single Nucleotide Polymorphisms in LIPAMEncoding

[0347] Polynucleotides

[0348] Common DNA sequence variants known as single nucleotidepolymorphisms (SNPs) were identified in SEQ ID NO:10-18 using theLIFESEQ database (Incyte Genomics). Sequences from the same gene wereclustered together and assembled as described in Example III, allowingthe identification of all sequence variants in the gene. An algorithmconsisting of a series of filters was used to distinguish SNPs fromother sequence variants. Preliminary filters removed the majority ofbasecall errors by requiring a minimum Phred quality score of 15, andremoved sequence alignment errors and errors resulting from impropertrimming of vector sequences, chimeras, and splice variants. Anautomated procedure of advanced chromosome analysis analysed theoriginal chromatogram files in the vicinity of the putative SNP. Cloneerror filters used statistically generated algorithms to identify errorsintroduced during laboratory processing, such as those caused by reversetranscriptase, polymerase, or somatic mutation. Clustering error filtersused statistically generated algorithms to identify errors resultingfrom clustering of close homologs or pseudogenes, or due tocontamination by non-human sequences. A final set of filters removedduplicates and SNPs found in immunoglobulins or T-cell receptors.

[0349] Certain SNPs were selected for further characterization by massspectrometry using the high throughput MASSARRAY system (Sequenom, Inc.)to analyze allele frequencies at the SNP sites in four different humanpopulations. The Caucasian population comprised 92 individuals (46 male,46 female), including 83 from Utah, four French, three Venezualan, andtwo Amish individuals. The African population comprised 194 individuals(97 male, 97 female), all African Americans. The Hispanic populationcomprised 324 individuals (162 male, 162 female), all Mexican Hispanic.The Asian population comprised 126 individuals (64 male, 62 female) witha reported parental breakdown of 43% Chinese, 31% Japanese, 13% Korean,5% Vietnamese, and 8% other Asian. Allele frequencies were firstanalyzed in the Caucasian population; in some cases those SNPs whichshowed no allelic variance in this population were not further tested inthe other three populations.

[0350] X. Labeling and Use of Individual Hybridization Probes

[0351] Hybridization probes derived from SEQ ID NO:10-18 are employed toscreen cDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 software (National Biosciences) and labeled bycombining 50 pmol of each oligomer, 250 μCi of [γ-³²P] adenosinetriphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase(DuPont NEN, Boston Mass.). The labeled oligonucleotides aresubstantially purified using a SEPHADEX G-25 superfine size exclusiondextran bead column (Amersham Pharmacia Biotech). An aliquot containing107 counts per minute of the labeled probe is used in a typicalmembrane-based hybridization analysis of human genomic DNA digested withone of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xba I,or Pvu II (DuPont NEN).

[0352] The DNA from each digest is fractionated on a 0.7% agarose geland transferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under conditions of up to, for example, 0.1× saline sodiumcitrate and 0.5% sodium dodecyl sulfate. Hybridization patterns arevisualized using autoradiography or an alternative imaging means andcompared.

[0353] XI. Microarrays

[0354] The linkage or synthesis of array elements upon a microarray canbe achieved utilizing photolithography, piezoelectric printing (ink-jetprinting, See, e.g., Baldeschweiler, supra.), mechanical microspottingtechnologies, and derivatives thereof. The substrate in each of theaforementioned technologies should be uniform and solid with anon-porous surface (Schena (1999), surra). Suggested substrates includesilicon, silica, glass slides, glass chips, and silicon wafers.Alternatively, a procedure analogous to a dot or slot blot may also beused to arrange and link elements to the surface of a substrate usingthermal, UV, chemical, or mechanical bonding procedures. A typical arraymay be produced using available methods and machines well known to thoseof ordinary skill in the art and may contain any appropriate number ofelements. (See, e.g., Schena, M. et al. (1995) Science 270:467470;Shalon, D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and J.Hodgson (1998) Nat. Biotechnol. 16:27-31.)

[0355] Full length cDNAs, Expressed Sequence Tags (ESTs), or fragmentsor oligomers thereof may comprise the elements of the microarray.Fragments or oligomers suitable for hybridization can be selected usingsoftware well known in the art such as LASERGENE software (DNASTAR). Thearray elements are hybridized with polynucleotides in a biologicalsample. The polynucleotides in the biological sample are conjugated to afluorescent label or other molecular tag for ease of detection. Afterhybridization, nonhybridized nucleotides from the biological sample areremoved, and a fluorescence scanner is used to detect hybridization ateach array element. Alternatively, laser desorbtion and massspectrometry may be used for detection of hybridization. The degree ofcomplementarity and the relative abundance of each polynucleotide whichhybridizes to an element on the microarray may be assessed. In oneembodiment, microarray preparation and usage is described in detailbelow.

[0356] Tissue or Cell Sample Preparation

[0357] Total RNA is isolated from tissue samples using the guanidiniumthiocyanate method and poly(A)⁺ RNA is purified using the oligo-(dT)cellulose method. Each poly(A)⁺ RNA sample is reverse transcribed usingMMLV reverse-transcriptase, 0.05 pg/μl oligo-(dT) primer (21mer), 1×first strand buffer, 0.03 units/μl RNase inhibitor, 500 μM dATP, 500 μMdGTP, 500 μM dTTP, 40 μM dCTP, 40 μM dCTP-Cy3 (BDS) or dCTP-Cy5(Amersham Pharmacia Biotech). The reverse transcription reaction isperformed in a 25 ml volume containing 200 ng poly(A)⁺ RNA withGEMBRIGHT kits (Incyte). Specific control poly(A)⁺ RNAs are synthesizedby in vitro transcription from non-coding yeast genomic DNA. Afterincubation at 37° C. for 2 hr, each reaction sample (one with Cy3 andanother with Cy5 labeling) is treated with 2.5 ml of 0.5M sodiumhydroxide and incubated for 20 minutes at 85° C. to the stop thereaction and degrade the RNA. Samples are purified using two successiveCHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc.(CLONTECH), Palo Alto Calif.) and after combining, both reaction samplesare ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodiumacetate, and 300 ml of 100% ethanol. The sample is then dried tocompletion using a SpeedVAC (Savant Instruments Inc., Holbrook N.Y.) andresuspended in 14 μl 5×SSC/0.2% SDS.

[0358] Microarray Preparation

[0359] Sequences of the present invention are used to generate arrayelements. Each array element is amplified from bacterial cellscontaining vectors with cloned cDNA inserts. PCR amplification usesprimers complementary to the vector sequences flanking the cDNA insert.Array elements are amplified in thirty cycles of PCR from an initialquantity of 1-2 ng to a final quantity greater than 5 μg. Amplifiedarray elements are then purified using SEPHACRYL400 (Amersham PharmaciaBiotech).

[0360] Purified array elements are immobilized on polymer-coated glassslides. Glass microscope slides (Coming) are cleaned by ultrasound in0.1% SDS and acetone, with extensive distilled water washes between andafter treatments. Glass slides are etched in 4% hydrofluoric acid (VWRScientific Products Corporation (VWR), West Chester Pa.), washedextensively in distilled water, and coated with 0.05% aminopropyl silane(Sigma) in 95% ethanol. Coated slides are cured in a 100° C. oven.

[0361] Array elements are applied to the coated glass substrate using aprocedure described in U.S. Pat. No. 5,807,522, incorporated herein byreference. 1 μl of the array element DNA, at an average concentration of100 ng/μl, is loaded into the open capillary printing element by ahigh-speed robotic apparatus. The apparatus then deposits about 5 nl ofarray element sample per slide.

[0362] Microarrays are UV-crosslinked using a STRATALINKERUV-crosslinker (Stratagene). Microarrays are washed at room temperatureonce in 0.2% SDS and three times in distilled water. Non-specificbinding sites are blocked by incubation of microarrays in 0.2% casein inphosphate buffered saline (PBS) (Tropix, Inc., Bedford Mass.) for 30minutes at 60° C. followed by washes in 0.2% SDS and distilled water asbefore.

[0363] Hybridization

[0364] Hybridization reactions contain 9 μl of sample mixture consistingof 0.2 μg each of Cy3 and Cy5 labeled cDNA synthesis products in 5×SSC,0.2% SDS hybridization buffer. The sample mixture is heated to 65° C.for 5 minutes and is aliquoted onto the microarray surface and coveredwith an 1.8 cm² coverslip. The arrays are transferred to a waterproofchamber having a cavity just slightly larger than a microscope slide.The chamber is kept at 100% humidity internally by the addition of 140μl of 5×SSC in a corner of the chamber. The chamber containing thearrays is incubated for about 6.5 hours at 60° C. The arrays are washedfor 10 min at 45° C. in a first wash buffer (1×SSC, 0.1% SDS), threetimes for 10 minutes each at 45° C. in a second wash buffer (0.1×SSC),and dried.

[0365] Detection

[0366] Reporter-labeled hybridization complexes are detected with amicroscope equipped with an Innova 70 mixed gas 10 W laser (Coherent,Inc., Santa Clara Calif.) capable of generating spectral lines at 488 nmfor excitation of Cy3 and at 632 nm for excitation of Cy5. Theexcitation laser light is focused on the array using a 20× microscopeobjective (Nikon, Inc., Melville N.Y.). The slide containing the arrayis placed on a computer-controlled X-Y stage on the microscope andraster-scanned past the objective. The 1.8 cm×1.8 cm array used in thepresent example is scanned with a resolution of 20 micrometers.

[0367] In two separate scans, a mixed gas multiline laser excites thetwo fluorophores sequentially. Emitted light is split, based onwavelength, into two photomultiplier tube detectors (PMT R1477,Hamamatsu Photonics Systems, Bridgewater N.J.) corresponding to the twofluorophores. Appropriate filters positioned between the array and thephotomultiplier tubes are used to filter the signals. The emissionmaxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5.Each array is typically scanned twice, one scan per fluorophore usingthe appropriate filters at the laser source, although the apparatus iscapable of recording the spectra from both fluorophores simultaneously.

[0368] The sensitivity of the scans is typically calibrated using thesignal intensity generated by a cDNA control species added to the samplemixture at a known concentration. A specific location on the arraycontains a complementary DNA sequence, allowing the intensity of thesignal at that location to be correlated with a weight ratio ofhybridizing species of 1:100,000. When two samples from differentsources (e.g., representing test and control cells), each labeled with adifferent fluorophore, are hybridized to a single array for the purposeof identifying genes that are differentially expressed, the calibrationis done by labeling samples of the calibrating cDNA with the twofluorophores and adding identical amounts of each to the hybridizationmixture.

[0369] The output of the photomultiplier tube is digitized using a12-bit RTI-835H analog-to-digital (A/D) conversion board (AnalogDevices, Inc., Norwood Mass.) installed in an IBM-compatible PCcomputer. The digitized data are displayed as an image where the signalintensity is mapped using a linear 20-color transformation to apseudocolor scale ranging from blue (low signal) to red (high signal).The data is also analyzed quantitatively. Where two differentfluorophores are excited and measured simultaneously, the data are firstcorrected for optical crosstalk (due to overlapping emission spectra)between the fluorophores using each fluorophore's emission spectrum.

[0370] A grid is superimposed over the fluorescence signal image suchthat the signal from each spot is centered in each element of the grid.The fluorescence signal within each element is then integrated to obtaina numerical value corresponding to the average intensity of the signal.The software used for signal analysis is the GEMTOOLS gene expressionanalysis program (Incyte).

[0371] For example, component 1824717_HGG4 of SEQ ID NO:15 showeddifferential expression in tissue affected by cancer versus normaltissue, as determined by microarray analysis. Matched samples of normallung tissue and lung tissue affected by squamous cell carcinoma, andmatched samples of normal lung tissue and lung tissue affected affectedby adenocarcinoma, were provided by the Roy Castle International Centerfor Lung Cancer Research (Liverpool, UK). The expression of component1824717_HGG4 was altered in lung tissue affected by squamous cellcarcinoma and in lung tissue affected by adenocarcinoma. Therefore, SEQID NO:15 is useful in diagnostic assays for cancer.

[0372] XII. Complementary Polynucleotides

[0373] Sequences complementary to the LIPAM-encoding sequences, or anyparts thereof, are used to detect, decrease, or inhibit expression ofnaturally occurring LIPAM. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using OLIGO 4.06 software(National Biosciences) and the coding sequence of LIPAM. To inhibittranscription, a complementary oligonucleotide is designed from the mostunique 5′ sequence and used to prevent promoter binding to the codingsequence. To inhibit translation, a complementary oligonucleotide isdesigned to prevent ribosomal binding to the LIPAM-encoding transcript.

[0374] XIII. Expression of LIPAM

[0375] Expression and purification of LIPAM is achieved using bacterialor virus-based expression systems. For expression of LIPAM in bacteria,cDNA is subcloned into an appropriate vector containing an antibioticresistance gene and an inducible promoter that directs high levels ofcDNA transcription. Examples of such promoters include, but are notlimited to, the trp-lac (tac) hybrid promoter and the T5 or T7bacteriophage promoter in conjunction with the lac operator regulatoryelement. Recombinant vectors are transformed into suitable bacterialhosts, e.g., BL21(DE3). Antibiotic resistant bacteria express LIPAM uponinduction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expressionof LIPAM in eukaryotic cells is achieved by infecting insect ormammalian cell lines with recombinant Autographica californica nuclearpolyhedrosis virus (AcMNPV), commonly known as baculovirus. Thenonessential polyhedrin gene of baculovirus is replaced with cDNAencoding LIPAM by either homologous recombination or bacterial-mediatedtransposition involving transfer plasmid intermediates. Viralinfectivity is maintained and the strong polyhedrin promoter drives highlevels of cDNA transcription. Recombinant baculovirus is used to infectSpodoptera frugiperda (Sf9) insect cells in most cases, or humanhepatocytes, in some cases. Infection of the latter requires additionalgenetic modifications to baculovirus. (See Engelhard, E. K. et al.(1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996)Hum. Gene Ther. 7:1937-1945.)

[0376] In most expression systems, LIPAM is synthesized as a fusionprotein with, e.g., glutathione S-transferase (GST) or a peptide epitopetag, such as FLAG or 6-His, permitting rapid, single-step,affinity-based purification of recombinant fusion protein from crudecell lysates. GST, a 26-kilodalton enzyme from Schistosoma japonicum,enables the purification of fusion proteins on immobilized glutathioneunder conditions that maintain protein activity and antigenicity(Amersham Pharmacia Biotech). Following purification, the GST moiety canbe proteolytically cleaved from LIPAM at specifically engineered sites.FLAG, an 8-amino acid peptide, enables immunoaffinity purification usingcommercially available monoclonal and polyclonal anti-FLAG antibodies(Eastman Kodak). 6-His, a stretch of six consecutive histidine residues,enables purification on metal-chelate resins (QIAGEN). Methods forprotein expression and purification are discussed in Ausubel (1995,supra, ch. 10 and 16). Purified LIPAM obtained by these methods can beused directly in the assays shown in Examples XVII and XVIII, whereapplicable.

[0377] XIV. Functional Assays

[0378] LIPAM function is assessed by expressing the sequences encodingLIPAM at physiologically elevated levels in mammalian cell culturesystems. cDNA is subcloned into a mammalian expression vector containinga strong promoter that drives high levels of cDNA expression. Vectors ofchoice include PCMV SPORT (Life Technologies) and PCR3.1 (Invitrogen,Carlsbad Calif.), both of which contain the cytomegalovirus promoter.5-10 μg of recombinant vector are transiently transfected into a humancell line, for example, an endothelial or hematopoietic cell line, usingeither liposome formulations or electroporation. 1-2 μg of an additionalplasmid containing sequences encoding a marker protein areco-transfected. Expression of a marker protein provides a means todistinguish transfected cells from nontransfected cells and is areliable predictor of cDNA expression from the recombinant vector.Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP;Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), anautomated, laser optics-based technique, is used to identify transfectedcells expressing GFP or CD64-GFP and to evaluate the apoptotic state ofthe cells and other cellular properties. FCM detects and quantifies theuptake of fluorescent molecules that diagnose events preceding orcoincident with cell death. These events include changes in nuclear DNAcontent as measured by staining of DNA with propidium iodide; changes incell size and granularity as measured by forward light scatter and 90degree side light scatter; down-regulation of DNA synthesis as measuredby decrease in bromodeoxyuridine uptake; alterations in expression ofcell surface and intracellular proteins as measured by reactivity withspecific antibodies; and alterations in plasma membrane composition asmeasured by the binding of fluorescein-conjugated Annexin V protein tothe cell surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994) Flow Cytometry, Oxford, New York N.Y.

[0379] The influence of LIPAM on gene expression can be assessed usinghighly purified populations of cells transfected with sequences encodingLIPAM and either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed onthe surface of transfected cells and bind to conserved regions of humanimmunoglobulin G (IgG). Transfected cells are efficiently separated fromnontransfected cells using magnetic beads coated with either human IgGor antibody against CD64 (DYNAL, Lake Success N.Y.). mRNA can bepurified from the cells using methods well known by those of skill inthe art. Expression of mRNA encoding LIPAM and other genes of interestcan be analyzed by northern analysis or microarray techniques.

[0380] XV. Production of LIPAM Specific Antibodies

[0381] LIPAM substantially purified using polyacrylamide gelelectrophoresis (PAGE; see, e.g., Harrington, M. G. (1990) MethodsEnzymol. 182:488-495), or other purification techniques, is used toimmunize animals (e.g., rabbits, mice, etc.) and to produce antibodiesusing standard protocols.

[0382] Alternatively, the LIPAM amino acid sequence is analyzed usingLASERGENE software (DNASTAR) to determine regions of highimmunogenicity, and a corresponding oligopeptide is synthesized and usedto raise antibodies by means known to those of skill in the art. Methodsfor selection of appropriate epitopes, such as those near the C-terminusor in hydrophilic regions are well described in the art. (See, e.g.,Ausubel, 1995, supra, ch. 11.)

[0383] Typically, oligopeptides of about 15 residues in length aresynthesized using an ABI 431A peptide synthesizer (Applied Biosystems)using FMOC chemistry and coupled to KLH (Sigma-Aldrich, St. Louis Mo.)by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) toincrease immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits areimmunized with the oligopeptide-KLH complex in complete Freund'sadjuvant. Resulting antisera are tested for antipeptide and anti-LIPAMactivity by, for example, binding the peptide or LIPAM to a substrate,blocking with 1% BSA, reacting with rabbit antisera, washing, andreacting with radio-iodinated goat anti-rabbit IgG.

[0384] XVI. Purification of Naturally Occurring LIPAM Using SpecificAntibodies

[0385] Naturally occurring or recombinant LIPAM is substantiallypurified by immunoaffinity chromatography using antibodies specific forLIPAM. An immunoaffinity column is constructed by covalently couplinganti-LIPAM antibody to an activated chromatographic resin, such asCNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After thecoupling, the resin is blocked and washed according to themanufacturer's instructions.

[0386] Media containing LIPAM are passed over the immunoaffinity column,and the column is washed under conditions that allow the preferentialabsorbance of LIPAM (e.g., high ionic strength buffers in the presenceof detergent). The column is eluted under conditions that disruptantibody/LIPAM binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), andLIPAM is collected.

[0387] XVII. Identification of Molecules which Interact with LIPAM

[0388] LIPAM, or biologically active fragments thereof, are labeled with¹²⁵I Bolton-Hunter reagent. (See, e.g., Bolton, A. E. and W. M. Hunter(1973) Biochem. J. 133:529-539.) Candidate molecules previously arrayedin the wells of a multi-well plate are incubated with the labeled LIPAM,washed, and any wells with labeled LIPAM complex are assayed. Dataobtained using different concentrations of LIPAM are used to calculatevalues for the number, affinity, and association of LIPAM with thecandidate molecules.

[0389] Alternatively, molecules interacting with LIPAM are analyzedusing the yeast two-hybrid system as described in Fields, S. and O. Song(1989) Nature 340:245-246, or using commercially available kits based onthe two-hybrid system, such as the MATCHMAKER system (Clontech).

[0390] LIPAM may also be used in the PATHCALLING process (CuraGen Corp.,New Haven Conn.) which employs the yeast two-hybrid system in ahigh-throughput manner to determine all interactions between theproteins ncoded by two large libraries of genes (Nandabalan, K. et al.(2000) U.S. Pat. No. 6,057,101).

[0391] XVIII. Demonstration of LIPAM Activity

[0392] Selected candidate lipid molecules, such as C4 sterols,oxysterol, apolipoprotein E, and phospholipids, are arrayed in the wellsof a multi-well plate. LIPAM, or biologically active fragments thereof,are labeled with ¹²⁵I Bolton-Hunter reagent. (See, e.g., Bolton A. E.and W. M. Hunter (1973) Biochem. J. 133:529-539.) The selected candidatelipid molecules are incubated with the labeled LIPAM and washed. Anywells with labeled LIPAM complex are assayed. Data obtained usingdifferent concentrations of LIPAM are used to calculate values for thenumber, affinity, and association of LIPAM with the candidate molecules.Significant binding of LIPAM to the candidate lipid molecules isindicative of LIPAM activity.

[0393] In the alternative, LIPAM activity is determined in a continuousfluorescent transfer assay using as substrate1-palmitoyl-2-pyrenyldecanoyl-phosphatidylinositol (Phy(10)PI). Theassay measures the increase of pyrene monomer fluorescence intensity asa result of the transfer of pyrenylacyl (Pyr(x))-labeled phospholipidfrom quenched donor vesicles to unquenched acceptor vesicles (VanParidon et al. (1988) Biochemistry 27:6208-6214). Donor vesicles consistof Pyr(x) phosphatidylinositol (Pyr(x)PI),2,4,6-trinitrophenylphosphatidylethanolamine (TNP-PE) and eggphosphatidylcholine (PC) in a mol % ratio of 10:10:80 (2 nmol of totalphospholipid). Acceptor vesicles consist of phosphatidic acid (PA) andegg PC in a mol % ratio of 5:95 (25-fold excess of total phospholipid).The reaction is carried out in 2 ml of 20 mM Tris-HCl, 5 mM EDTA, 200 mMNaCl (pH 7.4) containing 0.1 mg of BSA at 37° C. The reaction isinitiated by the addition of 10-50 μl of LIPAM. Measurements areperformed using a fluorimeter equipped with a thermostated cuvetteholder and a stirring device. The initial slope of the progress curve istaken as an arbitrary unit of transfer activity (van Tiel, C. M. et al.(2000) J. Biol. Chem. 275:21532-21538; Westerman, J. et al. (1995) J.Biol. Chem. 270:14263-14266).

[0394] In the alternative, LIPAM activity is determined by measuring therate of incorporation of a radioactive fatty acid precursor into fattyacyl-CoA. The final reaction contains 200 mM Tris-HCl, pH 7.5, 2.5 mMATP, 8 mM MgCl₂, 2 mM EDTA, 20 mM NaF, 0.1% Triton X-100, 10 mM[³H]oleate, [³H]myristate or [¹⁴C]decanoate, 0.5 mM coenzyme A, andLIPAM in a total volume of 0.5 ml. The reaction is initiated with theaddition of coenzyme A, incubated at 35° C. for 10 min, and terminatedby the addition of 2.5 ml of isopropyl alcohol, n-heptane, 1 M H₂SO₄(40:10:1). Radioactive fatty acid is removed by organic extraction usingn-heptane. Fatty acyl-CoA formed during the reaction remains in theaqueous fraction and is quantified by scintillation counting (Black, P.N. et al. (1997) J. Biol. Chem. 272: 4896-4904).

[0395] In the alternative, LIPAM activity is determined by measuring thedegradation of the sphingolipid glucosylceramide. 25-50 microunitsglucocerebrosidase are incubated with varying concentrations of LIPAM ina 40 μl reaction at 37° C. for 20 min. The final reaction contains 50 mMsodium citrate pH 4.5, 20 ng human serum albumin, and 3.125 mM lipids inthe form of liposomes, which contain lipids in the followingproportions: [¹⁴C]glucosylceramide (3 mol %, 2.4 Ci/mol), cholesterol(23 mol %), phosphatidic acid (20 mol %), phosphatidylcholine (54 mol%). The reaction is stopped by the addition of 160 μlchloroform/methanol (2:1) and 20 μl 0.1% glucose, and shaking. Aftercentrifugation at 4000 rpm, enzymatically released [¹⁴C]glucose in theaqueous phase is measured in a scintillation counter. LIPAM activity isdetermined by its effect on increasing the rate of glucosylceramidehydrolysis by glucocerebrosidase (Wilkening, G. et al. J. Biol. Chem.(1998) 273:30271-30278).

[0396] In the alternative, LIPAM activity can be demonstrated by an invitro hydrolysis assay with vesicles containing1-palmitoyl-2-[1-¹⁴C]oleoyl phosphatidylcholine (Sigma-Aldrich). LIPAMtriglyceride lipase activity and phospholipase A₂ activity aredemonstrated by analysis of the cleavage products isolated from thehydrolysis reaction mixture.

[0397] Vesicles containing 1-palmitoyl-2-[1-¹⁴C]oleoylphosphatidylcholine (Amersham Pharmacia Biotech.) are prepared by mixing2.0 μCi of the radiolabeled phospholipid with 12.5 mg of unlabeled1-palmitoyl-2-oleoyl phosphatidylcholine and drying the mixture underN₂. 2.5 ml of 150 mM Tris-HCl, pH 7.5, is added, and the mixture issonicated and centrifuged. The supernatant may be stored at 4° C. Thefinal reaction mixtures contain 0.25 ml of Hanks buffered salt solutionsupplemented with 2.0 mM taurochenodeoxycholate, 1.0% bovine serumalbumin, 1.0 mM CaCl₂, pH 7.4, 150 μg of 1-palmitoyl-2-[1-¹⁴C]oleoylphosphatidylcholine vesicles, and various amounts of LIPAM diluted inPBS. After incubation for 30 min at 37° C., 20 μg each oflyso-phosphatidylcholine and oleic acid are added as carriers and eachsample is extracted for total lipids. The lipids are separated by thinlayer chromatography using a two solvent system ofchloroform:methanol:acetic acid:water (65:35:8:4) until the solventfront is halfway up the plate. The process is then continued withhexane:ether:acetic acid (86:16:1) until the solvent front is at the topof the plate. The lipid-containing areas are visualized with I₂ vapor;the spots are scraped, and their radioactivity is determined byscintillation counting. The amount of radioactivity released as fattyacids will increase as a greater amount of LIPAM is added to the assaymixture while the amount of radioactivity released aslysophosphatidylcholine will remain low. This demonstrates that LIPAMcleaves at the sn-2 and not the sn-1 position, as is characteristic ofphospholipase A₂ activity.

[0398] In the alternative, phospholipase activity of LIPAM is measuredby the hydrolysis of a fatty acyl residue at the sn-1 position ofphosphatidylserine. LIPAM is combined with the tritium [³H] labeledsubstrate phosphatidylserine at stoichiometric quantities in a suitablebuffer. Following an appropriate incubation time, the hydrolyzedreaction products are separated from the substrates by chromatographicmethods. The amount of acylglycerophosphoserine produced is measured bycounting tritiated product with the help of a scintillation counter.Various control groups are set up to account for background noise andunincorporated substrate. The final counts represent the tritiatedenzyme product [³H]-acylglycerophosphoserine, which is directlyproportional to the activity of LIPAM in biological samples.

[0399] Lipoxygenase activity of LIPAM can be measured by chromatographicmethods. Extracted LIPAM lipoxygenase protein is incubated with 100 μM[1-¹⁴C] arachidonic acid or other unlabeled fatty acids at 37° C. for 30min. After the incubation, stop solution (acetonitrile:methanol:water,350:150:1) is added. The samples are extracted and analyzed byreverse-phase HPLC using a solvent system of methano/water/acetic acid,85:15:0.01 (vol/vol) at a flow rate of 1 ml/min. The effluent ismonitored at 235 nm and analyzed for the presence of the majorarachidonic metabolite such as 12-HPETE (catalyzed by 12-LOX). Thefractions are also subjected to liquid scintillation counting. The finalcounts represent the products, which is directly proportional to theactivity of LIPAM in biological samples. For stereochemical analysis,the metabolites of arachidonic acid are analyzed further by chiralphase-HPLC and by mass spectrometry (Sun, D. et al. (1998) J. Biol.Chem. 273:33540-33547).

[0400] Sialidase activity of LIPAM is assayed using various substrates,including but not limited to2′-(4-methylumbelliferyl)α-D-N-acetylneuramic acid,2′-O-(o-nitrophenyl)α-D-N-acetylneuramic acid,2′-O-(p-nitrophenyl)α-D-N-acetylneuramic acid, and α(2-3)- andα(2-6)-sialyllactose. The reaction mixture contains 30 nmol substrate,0.2 mg bovine serum albumin, 10 μmol sodium acetate (pH 4.6), 0.2 mgTriton X-100, and purified LIPAM (or a sample containing LIPAM).Following incubation at 37° C. for 10-30 min, the released sialic acidis quantified using the thiobarbituric acid method (Aminoff, D. (1961)Biochem. J. 81:384-392). One unit of sialidase activity is defined asthe amount of LIPAM that catalyzes the release of 1 nmol of sialic acidfrom substrate per hour (Hasegawa, T. et al. (2000) J. Biol. Chem.275:8007-8015).

[0401] Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with certain embodiments,it should be understood that the invention as claimed should not beunduly limited to such specific embodiments. Indeed, variousmodifications of the described modes for carrying out the inventionwhich are obvious to those skilled in molecular biology or relatedfields are intended to be within the scope of the following claims.TABLE 1 Poly- peptide Incyte SEQ ID Incyte Polynucleotide Incyte ProjectID NO: Polypeptide ID SEQ ID NO: Polynucleotide ID 7472774 1 7472774CD110 7472774CB1 2884821 2 2884821CD1 11 2884821CB1 72852842 3 72852842CD112 72852842CB1 7484271 4 7484271CD1 13 7484271CB1 7474074 5 7474074CD114 7474074CB1 72024970 6 72024970CD1 15 72024970CB1 6131380 7 6131380CD116 6131380CB1 643681 8 643681CD1 17 643681CB1 6897474 9 6897474CD1 186897474CB1

[0402] TABLE 2 Incyte Polypeptide Polypeptide GenBank ProbabilityGenBank SEQ ID NO: ID ID NO: Score Homolog 1 7472774CD1 g48869781.6E−161 [Homo sapiens] cytosolic phospholipase A2 beta; cPLA2beta(Song, C. et al. (1999) J. Biol. Chem. 274: 17063-17067) 2 2884821CD1g14669826 0.0 lipoic acid synthase [Mus musculus] (Morikawa, T. et al.(2001) FEBS Lett. 498: 16-21) 3 72852842CD1 g4894788 2.5E−126 [Musmusculus] phospholipase C delta-1 (Lee, W. K. et al. (1999) Biochem.Biophys. Res. Commun. 261: 393-399) 4 7484271CD1 g2138183 2.7E−11 [Musmusculus] polycystic kidney disease 1 protein (Lohning, C. et al. (1997)Mamm. Genome 8: 307-311) 5 7474074CD1 g4090960 3.1E−63 [Homo sapiens]phosphatidylserine-specific phospholipase A1 (Nagai, Y. et al. (1999) J.Biol. Chem. 274: 11053-11059) g13560884 1.0E-109 lacrimal lipase[Oryctolagus cuniculus] 6 72024970CD1 g6705987 1.1E−164 [Mus musculus]phospholipase C-L2 (Otsuki, M. (1999) Biochem. Biophys. Res. Commun.266: 97-103) 7 6131380CD1 g5771350 0.0 [Mus musculus] M-RdgB2 retinaldegeneration protein B subtype 2 (Lu, C. et al. (1999) J. Neurosci. 19:7317-7325) g2618983 0.0 [Mus musculus] membrane-associatedphosphatidylinositol transfer protein (Aikawa, Y. et al. (1997) Biochem.Biophys. Res. Commun. 236: 559-564) 8 643681CD1 g8452870 1.2E−36 [Homosapiens] lipopolysaccharide specific response-68 protein 9 6897474CD1g6651241 3.8E−188 [Mus musculus] TAGL-beta

[0403] TABLE 3 Amino SEQ Incyte Acid Potential Potential Analytical IDPolypeptide Resi- Phosphoryla- Glycosyla- Signature Sequences, Methodsand NO: ID dues tion Sites tion Sites Domains and Motifs Databases 17472774CD1 996 S34 S46 S64 N201 N362 Transmembrane domains: TMAP S133S151 N718 N834 E276-L301, S696-S720 S169 S183 N914 N terminus iscytosolic S219 S273 CYTOSOLIC PHOSPHOLIPASE A2 CPLA2 BLAST- S294 S418INCLUDES: PHOSPHATIDYLCHOLINE 2 PRODOM S557 S652 ACYLHYDROLASELYSOPHOSPHOLIPASE S662 S769 HYDROLASE LIPID S928 T17 T24 PD014471:G542-L711, G812-N914 T87 T104 T227 T248 T368 T603 T722 T775 T808 T916T952 Y38 Y664 2 2884821CD1 372 S2 S30 S57 SYNTHETASE LIPOIC ACID LIPSYNLIPOATE BLAST- S145 S229 IRONSULFUR SYNTHASE PRECURSOR PRODOM S258 S352MITOCHONDRION TRANSIT: T58 T96 T104 PD149846: L80-D135 T148 T163PD005028: Q311-E357 T178 T240 do SYNTHETASE; LIPOIC; ACID; BLAST-DOMOT292 T313 BIOSYNTHESIS: T369 DM02726|P32875|79-413: K36-K370DM02726|E36953|1-310: L74-A355 DM02726|G64043|7-320: L74-A355DM02726|P25845|7-320: N65-A355 Cell attachment sequence: R217-D219MOTIFS 3 72852842CD1 649 S313 S419 N417 N578 C2 domain: L525-T613HMMER-PFAM S429 S542 Phosphatidylinositol-specific HMMER-PFAM S574 T24T56 phospholipase C, Xdomain: T68 T79 T220 D156-K300 T267 T303 3 T381T397 Phosphatidylinositol-specific HMMER-PFAM T440 T452 phospholipase C,Y domain: A389-R506 T526 Phosphatidylinositol-specific BLIMPS-phospholipase signature BL50007: BLOCKS L161-G206, T220-Q257, L284-K300,H439-G480, Q600-L636 Phospholipase C signature PR00390: BLIMPS-P160-Q178, W186-G206, T283-K300, PRINTS I444-W465, W465-M483, L614-R624PHOSPHOLIPASE C PHOSPHODIESTERASE BLAST- HYDROLASE1PHOSPHATIDYLINOSITOL4 PRODOM 5BISPHOSPHATE LIPID DEGRADATION TRANSDUCERPHOSPHOINOSITIDESPECIFIC: PD001214: D156-K300 PD001202: L390-R506PHOSPHOLIPASE 1PHOSPHATIDYLINOSITOL4 BLAST- 5BISPHOSPHATEPHOSPHODIESTERASE PRODOM HYDROLASE LIPID DEGRADATION TRANSDUCER CCALCIUMBINDING: PD004439: R4-Q155 1-PHOSPHATIDYLINOSITOL-4,5- BLAST-DOMOBISPHOSPHATE PHOSPHODIESTERASE D: DM00855|P51178|64-472: W5-E332DM00712|P51178|474-754: K378-V645 DM00855|A48047|58-521: N47-S350DM00855|P10894|62-503: N47-D328 4 7484271CD1 2020 S76 S163 N341 N1128PLAT/LH2 (Polycystin-1, Lipoxygenase, HMMER-PFAM S245 S436 N1248Alpha-Toxin/lipoxygenase homology) S578 S598 N1296 domain: N769-E885,T1632-F1749, S693 S705 N1336 F431-Y550, T922-L1039, V1901-L2016, S706S710 N1533 I43-Y159, V1207-R1322, A300-L419, S711 S712 N1553V1505-C1620, V561-E677, F172-M286, S809 S812 N1680 V1053-L1177,I1374-R1489, T1763-E1883 4 S960 S965 N1790 Transmembrane domains: TMAPS1139 S1167 N1799 L31-D53, A1071-R1095, T1487-W1512 S1192 S1312 N1988N-terminus is non-cytosolic S1339 S1349 PROTEIN POLYCYSTIC KIDNEYDISEASE BLAST- S1406 S1424 REPEAT TRANSMEMBRANE POLYCYSTIN PRODOM S1535S1578 PRECURSOR AUTOSOMAL DOMINANT: S1607 S1715 PD010179: Y1634-K1793S1723 S1737 Cell attachment sequence: MOTIFS S1739 S1800 R1482-D1484,R2007-D2009 T16 T292 ATP/GTP-binding site motif A (P- MOTIFS T360 T427loop): G1239-S1246 T462 T656 T927 T949 T963 T996 T1092 T1166 T1173 T1184T1250 T1298 T1382 T1487 T1745 T1763 T1810 T1852 T1854 T1909 T1932 T1950T2012 Y771 Y790 Y1180 Y1329 Y1429 Y1975 5 7474074CD1 415 S145 S151 N18N351 signal cleavage: M1-S43 SPSCAN S232 S311 Lipase: M1-L275 HMMER-PFAMT27 T84 T135 Transmembrane domains: TMAP T304 T371 D106-I134, T278-M306T411 N terminus is non-cytosolic 5 Lipases, serine proteins BL00120:BLIMPS N62-I76, D106-S120, Y183-C193 BLOCKS Triacylglycerol lipasefamily BLIMPS- signature PR00821: N107-K125, PRINTS C206-T221, N19-Y38,I64-R79 Vespid venom allergen phospholipase BLIMPS- A1 signaturePR00825: PRINTS P148-H165, L171-P191 LIPASE PRECURSOR SIGNAL HYDROLASEBLAST- LIPID DEGRADATION GLYCOPROTEIN PRODOM PANCREATIC PROTEINPANCREAS: PD001492: N6-L314 TRIACYLGLYCEROL LIPASE: BLAST-DOMODM00344|A49488|25-326: M1-F294 DM00344|P11150|38-356: M1-L296DM00344|S15893|37-357: F25-F294 DM00344|P11153|17-335: N22-M270 672024970CD1 1152 S79 S91 S124 N290 N303 C2 domain: L756-T848 HMMER-PFAMS154 S186 N472 N534 PH domain: A44-A151 HMMER-PFAM S235 S276Phosphatidylinositol-specific HMMER-PFAM S350 S445 phospholipase:D323-K468, A621-C736 S483 S487 EF hand: W169-L197, R205-M234 HMMER-PFAMS543 S550 Phosphatidylinositol-specific BLIMPS- S558 S565 phospholipaseX-box domain protein BLOCKS S577 S584 BL50007: F669-G710, D835-I871,S591 S649 L328-G373, T387-Q424, L452-K468 S681 S881 C2 domain signaturePR00360: BLIMPS- S918 S932 R777-I789, N807-M820, V829-D837 PRINTS S980S1100 Phospholipase C signature PR00390: BLIMPS- S1111 T134 P327-Q345,D353-G373, T451-K468, PRINTS T173 T236 L674-W695, W695-L713, L849-R859T387 T504 T512 T615 6 T812 Y628 PHOSPHOLIPASE C PHOSPHODIESTERASE BLAST-HYDROLASE 1PHOSPHATIDYLINOSITOL4 PRODOM 5BISPHOSPHATE LIPID DEGRADATIONTRANSDUCER PHOSPHOINOSITIDE-SPECIFIC: PD001214: D323-K468 PD001202:L622-P732 PHOSPHOLIPASE 1PHOSPHATIDYLINOSITOL4 BLAST- 5BISPHOSPHATEPHOSPHODIESTERASE PRODOM HYDROLASE LIPID DEGRADATION TRANSDUCER CCALCIUM-BINDING: PD004439: Q100-Q322 1-PHOSPHATIDYLINOSITOL-4,5-BIS-BLAST-DOMO PHOSPHATE PHOSPHODIESTERASE D DM00855: P51178|64-472:S88-D494 P08487|71-500: I87-G500 P16885|63-486: I87-E485 P40977|208-616:I89-N499 EF-hand calcium-binding domain: MOTIFS D178-V190 7 6131380CD11294 S29 S38 S129 N31 N652 Phosphatidylinositol transfer HMMER-PFAM S164S205 N911 N945 protein: M1-L253 S254 S290 N1006 Transmembrane domain:G553-C572, TMAP S307 S310 A711-K737, T1050-V1065 S313 S314 N-terminus isnon-cytosolic S324 S340 Phosphatidylinositol transfer protein BLIMPS-S341 S353 signature PR00391: F198-D213, V219- PRINTS S367 S399 S238,E16-G35, V85-E105, I111-F126 S443 S444 PROTEIN PHOSPHATIDYLINOSITOLTRANSFER BLAST- S496 S504 ISOFORM PTD INS PTD INSTP LIPID PRODOM S584S585 BINDING TRANSPORT ALPHA PIT P ALPHA: S589 S592 PD006368: M1-M257 7S633 PROTEIN RETINAL DEGENERATION B BLAST- S637S644 PHOSPHATIDYLINOSITOLMEMBRANE PRODOM S666 S669 ASSOCIATED HOMOLOGUE OF DROSPHILA S767 S833GENE S891 S913 PD018514: P786-K1048 S1008 S1018 PD025569: G1049-L1200S1121 S1155 PD018515: R396-I565 S1203 S1222 PHOSPHATIDYLINOSITOL; BETA;BLAST_DOMO S1250 S1258 DEGENERATION; TRANSFER; DM02192: T13 T33 T59P43125|1-222: M1-D213 T155 T167 Q00169|1-213: M1-H212 T280 T433P53812|1-213: M1-I211, A850-D867 T704 T799 JX0316|1-214: M1-I211,A850-D867 T939 T966 Leucine zipper pattern: L1218-L1239 MOTIFS T1014T1037 Cell attachment sequence: MOTIFS T1096 T1179 R1033-D1035 T1282Y671 Y871 Y1090 8 643681CD1 77 T31 T32 T41 Signal cleavage: M1-P67SPSCAN 9 6897474CD1 576 S202 S239 N77 N367 Signal cleavage: M1-A21SPSCAN S558 S561 N485 Signal Peptide: M1-S22 HMMER T79 T154Transmembrane domains: TMAP T181 T213 V215-G234, P255-G283 T259 T498N-terminus is cytosolic T548 PROTEIN PEPTIDOGLYCAN RECOGNITION BLAST-PRECURSOR SIGNAL TUMOR ASSOCIATED CSP PRODOM PD090970: A368-Y486

[0404] TABLE 4 Polynucleotide SEQ ID NO:/ Incyte ID/ Sequence LengthSequence Fragments 10/7472774CB1/ 1-326, 1-558, 310-558, 463-2162,665-811, 1490-2112, 1491-1672, 1491-1787, 1491-1823, 3879 1491-1889,1491-1892, 1491-1958, 1491-1972, 1491-2022, 1491-2025, 1491-2119,1491-2120, 1493-2120, 1496-2120, 1497-2050, 1499-2120, 1557-2297,1587-1985, 1648-2120, 1675-2297, 1767-2120, 1807-2166, 1854-2297,1857-2297, 1926-2297, 1944-2150, 1944-3300, 2277-2949, 2281-2524,2281-2898, 2281-2949, 2282-2947, 2325-2934, 2330-2935, 2340-2935,2347-2934, 2387-2910, 2389-2934, 2395-2949, 2417-2524, 2445-2935,2464-2934, 2466-2934, 2488-2934, 2503-2935, 2514-2935, 2532-2934,2563-2935, 2597-2935, 2729-2878, 2729-3192, 2729-3249, 2729-3339,2729-3399, 2868-3420, 2934-3473, 2988-3592, 3066-3823, 3121-3726,3179-3879, 3182-3875, 3182-3876, 3182-3878, 3182-3879, 3183-3879,3184-3879, 3199-3565, 3220-3549, 3228-3802, 3269-3410, 3269-3646,3291-3879 11/2884821CB1/ 1-318, 52-352, 60-246, 60-318, 60-362, 60-371,60-743, 61-307, 63-314, 63-341, 81-391, 1623 88-366, 96-371, 99-725,107-351, 111-803, 124-380, 168-791, 231-672, 259-905, 263-932, 368-636,381-610, 381-845, 389-658, 389-833, 420-1067, 471-698, 524-1054,654-1087, 681-1236, 721-1255, 765-1317, 770-1271, 838-1233, 856-1078,856-1245, 856-1321, 856-1512, 879-1524, 952-1152, 1021-1496, 1050-1623,1076-1449, 1089-1623, 1136-1313, 1153-1595, 1182-1333 12/72852842CB1/1-445, 1-550, 1-853, 336-1614, 1197-1839, 1200-1840, 1249-1569,1249-1573, 1251-1569, 2199 1251-1573, 1288-1569, 1288-1573, 1300-2058,1336-2135, 1343-1818, 1383-2137, 1551-1814, 1551-2164, 1577-2137,1683-1835, 1727-2137, 1734-2197, 1758-2198, 1765-2197, 1768-2137,1769-2197, 1791-2137, 1798-2137, 1851-2137, 2004-2137, 2004-2199,2042-2137, 2058-2197 13/7484271CB1/ 1-130, 1-131, 1-341, 1-522, 1-712,22-131, 35-131, 36-129, 67-679, 69-91, 70-91, 129-149, 6326 129-150,158-285, 158-288, 158-527, 158-652, 158-672, 179-648, 192-858, 193-684,226-248, 227-248, 234-1071, 264-1071, 443-1071, 451-1071, 456-1070,523-1071, 535-1071, 558-1071, 565-1071, 568-1071, 574-1071, 577-1071,591-1071, 869-1267, 1177-1440, 1177-1699, 1321-2700, 1373-2040,1374-1821, 1374-2040, 1377-1998, 1377-2040, 1613-1935, 1884-2040,1889-2040, 1894-2040, 2061-2421, 2061-2434, 2061-2504, 2061-2553,2061-2570, 2061-2576, 2061-2628, 2061-2671, 2061-2674, 2061-2738,2061-2812, 2062-2253, 2062-2448, 2062-2572, 2062-2590, 2062-2672,2062-2673, 2062-2687, 2066-2639, 2129-2891, 2140-2639, 2145-2646,2149-2674, 2152-2768, 2155-2674, 2156-2783, 2160-2778, 2196-2930,2198-2918, 2212-2513, 2212-2639, 2212-2643, 2212-2666, 2212-2670,2212-2671, 2212-2683, 2212-2689, 2212-2700, 2218-2700, 2233-2904,2234-2952, 2249-2700, 2251-2907, 2251-2912, 2266-2700, 2493-2964,2494-2946, 2538-2700, 2553-2700, 2608-2700, 2691-3263, 2738-3245,2741-3238, 2741-3263, 2741-3266, 2741-3273, 2741-3326, 2837-3346,2837-3362, 2837-3368, 2837-3451, 2865-4748, 2870-3645, 2887-3575,2893-3405, 2913-3195, 2916-3489, 2921-3648, 2926-3548, 2932-3332,2933-3642, 2937-3271, 2938-3648, 2943-3648, 2952-3648, 2975-3648,2977-3342, 2978-3439, 2986-3640, 2989-3648, 2999-3648, 3012-3648,3031-3648, 3032-3551, 3033-3606, 3037-3648, 3044-3648, 3059-3648,3063-3641, 3074-3646, 3092-3648, 3103-3648, 3107-3644, 3111-3648,3117-3648, 3125-3642, 3128-3648, 3132-3648, 3137-3648, 3141-3648,3145-3648, 3151-3648, 3157-3648, 3158-3648, 3159-3648, 3193-3648,3211-3648, 3249-3648, 3319-3648, 3479-3648, 3653-3951, 4338-4616,4338-4865, 4397-4660, 4397-4762, 4397-5019, 4464-5142, 4542-5043,4633-5264, 4658-5249, 4722-5199, 4723-5268, 4773-5280, 4798-5285,4867-5400, 4890-5522, 4967-5582, 5024-5613, 5045-5079, 5045-5083,5045-5084, 5045-5091, 5048-5706, 5158-5713, 5178-5779, 5214-5890,5240-5889, 5269-5815, 5275-5892, 5290-5929, 5363-5620, 5373-5951,5381-5924, 5397-5747, 5478-6326, 5539-5915, 5801-6246, 5801-6298,5801-6326, 5833-6326, 5852-5898, 5855-5898, 5856-5898 14/7474074CB1/1-262, 132-1114, 260-322, 646-763, 646-771, 646-897, 718-771, 718-856,718-857, 718-859, 1561 718-860, 770-1161, 1099-1561, 1100-1290,1100-1556, 1100-1561, 1165-1561, 1273-1561, 1290-1559, 1302-1559,1395-1556 15/72024970CB1/ 1-698, 1-722, 1-740, 1-744, 1-753, 1-762,1-764, 1-818, 5-506, 109-686, 123-681, 160-756, 4941 212-870, 281-804,299-824, 338-862, 389-615, 418-1240, 426-1240, 428-822, 430-885,431-1240, 435-1019, 443-1240, 445-1094, 445-1107, 448-1240, 451-1138,454-1000, 454-1240, 456-1240, 463-1056, 470-1019, 471-1019, 471-1240,488-646, 488-1191, 488-1217, 488-1223, 488-1236, 488-1240, 491-1240,511-1008, 516-958, 520-979, 532-1240, 534-1240, 538-1166, 541-1240,548-1132, 571-1239, 578-1240, 582-1153, 607-1011, 607-1240, 622-1094,626-1240, 634-1240, 637-1240, 639-666, 641-686, 645-1240, 654-946,654-1198, 654-1199, 657-1240, 663-1240, 668-1240, 670-1240, 675-1240,676-1240, 677-761, 682-1240, 684-1240, 685-1240, 696-1240, 703-1240,728-1240, 736-1240, 737-1166, 744-1084, 747-1554, 754-1240, 798-1240,807-1240, 812-1240, 823-1240, 831-1107, 852-1240, 854-1240, 873-1239,873-1240, 877-1240, 903-1240, 909-1240, 910-1554, 912-1240, 913-1240,915-1240, 928-1240, 941-1240, 949-1077, 949-1379, 949-1418, 949-1423,949-1437, 949-1462, 949-1463, 949-1469, 949-1473, 949-1493, 950-1493,951-1493, 955-1493, 956-1493, 982-1240, 982-1487, 982-1497, 988-1012,988-1015, 988-1016, 990-1015, 994-1229, 994-1236, 994-1240, 996-1240,996-1565, 1031-1493, 1090-1631, 1090-1668, 1090-1670, 1096-1635,1096-1689, 1100-1630, 1119-1548, 1134-1665, 1134-1698, 1134-1734,1157-1740, 1158-1687, 1158-1689, 1165-1771, 1237-1493, 1259-1391,1275-1605, 1276-1493, 1347-1933, 1358-1896, 1369-1844, 1369-1860,1369-1865, 1369-1896, 1369-1900, 1369-1977, 1374-1495, 1380-1890,1404-1874, 1499-2026, 1499-2036, 1508-1976, 1524-2192, 1526-2192,1539-2050, 1554-2192, 1569-2193, 1572-2026, 1593-2192, 1594-2192,1604-2192, 1605-2191, 1609-2192, 1620-2192, 1624-2192, 1632-2192,1637-2192, 1652-2191, 1652-2192, 1666-2192, 1671-2192, 1735-1784,1735-2192, 1747-2192, 1757-2192, 1764-2192, 1767-2192, 1776-2261,1790-2406, 1839-2192, 1872-2192, 1888-2192, 1895-2192, 1916-2191,1916-2192, 1922-2192, 1936-2192, 1947-2192, 1998-2617, 2062-2625,2093-2192, 2094-2193, 2128-2434, 2128-2749, 2132-2291, 2136-2754,2199-2736, 2238-2980, 2287-2969, 2294-2558, 2303-2885, 2304-2980,2309-2759, 2309-2960, 2356-3100, 2371-2989, 2456-3077, 2491-2945,2540-2808, 2610-3047, 2611-3153, 2648-3156, 2668-2880, 2672-3326,2787-3622, 2810-3135, 2810-3139, 2813-3383, 2814-3244, 2814-3267,2814-3360, 2814-3376, 2814-3377, 2819-3376, 2823-3321, 2824-3374,2863-3217, 2883-3218, 2956-3377, 2996-3555, 3048-3803, 3092-3718,3113-3657, 3120-3215, 3205-3489, 3267-3485, 3355-3718, 3485-3715,3505-3718, 3539-3739, 3548-3718, 3551-3987, 3566-3716, 3610-3912,3721-3875, 3744-3799, 3745-4088, 3746-4083, 3759-4011, 3820-4344,3829-4078, 3849-3997, 3939-4509, 4064-4284, 4152-4632, 4213-4468,4213-4736, 4213-4761, 4213-4868, 4213-4941, 4227-4386, 4239-4468,4247-4744, 4262-4543 16/6131380CB1/ 1-353, 54-579, 293-934, 349-981,351-522, 376-643, 460-708, 488-809, 545-690, 667-1226, 4159 704-1358,805-1358, 911-1358, 1026-1226, 1030-1717, 1037-1467, 1039-1467,1044-1598, 1103-1714, 1119-1714, 1133-1717, 1316-1985, 1322-1753,1362-1691, 1497-1657, 1575-1985, 1715-2268, 1743-1938, 1766-2201,1771-2095, 1835-2332, 1885-2434, 1975-2329, 1993-2632, 2003-2507,2054-2991, 2267-2507, 2326-2507, 2330-2507, 2451-2507, 2888-415917/643681CB1/ 1-299, 51-328, 51-335, 84-353, 92-342, 95-315, 107-370,128-376, 139-459, 168-431, 181-447, 1481 191-421, 196-606, 217-516,287-520, 335-578, 344-599, 344-880, 361-659, 379-637, 385-617, 458-745,541-768, 541-805, 544-788, 544-1065, 681-1250, 708-939, 718-1299,726-969, 747-1399, 757-965, 772-1377, 794-1077, 796-1399, 811-1057,846-1394, 864-1120, 865-1127, 871-1107, 871-1341, 871-1399, 873-1402,904-1142, 904-1169, 975-1214, 1003-1242, 1021-1286, 1057-1245,1098-1281, 1112-1365, 1173-1391, 1173-1398, 1173-1427, 1173-1430,1237-1481 18/6897474CB1/ 1-534, 1-1841, 6-150, 7-196, 43-379, 43-572,59-584, 228-744, 228-810, 515-1169, 805-1409, 1841 889-1436, 889-1455,1216-1766, 1287-1834, 1486-1734, 1526-1836, 1526-1841

[0405] TABLE 5 Polynucleotide Incyte Representative SEQ ID NO: ProjectID Library 10 7472774CB1 MYEPTXT02 11 2884821CB1 TLYMNOT08 1272852842CB1 TESTNOT17 13 7484271CB1 BONRFEC01 14 7474074CB1 UTRSNOR01 1572024970CB1 LIVRTXS02 16 6131380CB1 NERDTDN03 17 643681CB1 PENITUT01 186897474CB1 LIVRTMR01

[0406] TABLE 6 Library Vector Library Description BONRFEC01 pINCY Thislarge size-fractionated library was constructed using RNA isolated fromrib bone tissue removed from a Caucasian male fetus who died fromPatau's syndrome (trisomy 13) at 20-weeks' gestation. Serologies werenegative. LIVRTMR01 PCDNA2.1 This random primed library was constructedusing RNA isolated from liver tissue removed from a 62-year-oldCaucasian female during partial hepatectomy and exploratory laparotomy.Pathology for the matched tumor tissue indicated metastatic intermediategrade neuroendocrine carcinoma, consistent with islet cell tumor,forming nodules ranging in size, in the lateral and medial left liverlobe. The pancreas showed fibrosis, chronic inflammation and fatnecrosis consistent with pseudocyst. The gallbladder showed mild chroniccholecystitis. Patient history included malignant neoplasm of thepancreas tail, pulmonary embolism, hyperlipidemia, thrombophlebitis,joint pain in multiple joints, type II diabetes, benign hypertension,cerebrovascular disease, and normal delivery. Previous surgeriesincluded distal pancreatectomy, total splenectomy, and partialhepatectomy. Family history included pancreas cancer with secondaryliver cancer, benign hypertension, and hyperlipidemia. LIVRTXS02 pINCYThis subtracted C3A liver tumor cell line tissue library was constructedusing 6.4 million clones from a treated C3A hepatocyte cell line libraryand was subjected to two rounds of subtraction hybridization with 1.72million clones from an untreated C3A hepatocyte cell line library. Thestarting library for subtraction was constructed using RNA isolated froma treated C3A hepatocyte cell line which is a derivative of Hep G2, acell line derived from a hepatoblastoma removed from a 15- year-oldCaucasian male. The cells were treated with 3-methylcholanthrene (MCA).The hybridization probe for subtraction was derived from a similarlyconstructed library from RNA isolated from untreated C3A hepatocytecells tissue from the same cell line. Subtractive hybridizationconditions were based on the methodologies of Swaroop, et al., NAR 19(1991): 1954 and Bonaldo, et al. Genome Research 6 (1996): 791.0MYEPTXT02 pINCY The library was constructed using RNA isolated from atreated K-562 cell line, derived from chronic myelogenous leukemiaprecursor cells removed from a 53-year-old female. The cells weretreated with 1 micromolar PMA for 96 hours. NERDTDN03 pINCY Thisnormalized dorsal root ganglion tissue library was constructed from 1.05million independent clones from a dorsal root ganglion tissue library.Starting RNA was made from dorsal root ganglion tissue removed from thecervical spine of a 32-year-old Caucasian male who died from acutepulmonary edema, acute bronchopneumonia, bilateral pleural effusions,pericardial effusion, and malignant lymphoma (natural killer cell type).The patient presented with pyrexia of unknown origin, malaise, fatigue,and gastrointestinal bleeding. Patient history included probablecytomegalovirus infection, liver congestion, and steatosis,splenomegaly, hemorrhagic cystitis, thyroid hemorrhage, respiratoryfailure, pneumonia of the left lung, natural killer cell lymphoma of thepharynx, Bell's palsy, and tobacco and alcohol abuse. Previous surgeriesincluded colonoscopy, closed colon biopsy, adenotonsillectomy, andnasopharyngeal endoscopy and biopsy. Patient medications includedDiflucan (fluconazole), Deltasone (prednisone), hydrocodone, Lortab,Alprazolam, Reazodone, ProMace-Cytabom, Etoposide, Cisplatin,Cytarabine, and dexamethasone. The patient received radiation therapyand multiple blood transfusions. The library was normalized in 2 roundsusing conditions adapted from Soares et al., PNAS (1994) 91: 9228-9232and Bonaldo et al., Genome Research 6 (1996): 791, except that asignificantly longer (48 hours/round) reannealing hybridization wasused. PENITUT01 pINCY Library was constructed using RNA isolated fromtumor tissue removed from the penis of a 64-year-old Caucasian maleduring penile amputation. Pathology indicated a fungating invasive grade4 squamous cell carcinoma involving the inner wall of the foreskin andextending onto the glans penis. Patient history included benign neoplasmof the large bowel, atherosclerotic coronary artery disease, anginapectoris, gout, and obesity. Family history included malignantpharyngeal neoplasm, chronic lymphocytic leukemia, and chronic liverdisease. TESTNOT17 pINCY Library was constructed from testis tissueremoved from a 26-year-old Caucasian male who died from head trauma dueto a motor vehicle accident. Serologies were negative. Patient historyincluded a hernia at birth, tobacco use (1 1/2 ppd), marijuana use, anddaily alcohol use (beer and hard liquor). TLYMNOT08 pINCY The librarywas constructed using RNA isolated from anergicallogenic T-lymphocytetissue removed from an adult (40-50-year-old) Caucasian male.The cellswere incubated for 3 days in the presence of 1 microgram/ml OKT3 mAb and5% human serum. UTRSNOR01 pINCY Library was constructed using RNAisolated from uterine endometrium tissue removed from a 29-year-oldCaucasian female during a vaginal hysterectomy and cystocele repair.Pathology indicated the endometrium was secretory, and the cervix showedmild chronic cervicitis with focal squamous metaplasia. Pathology forthe associated tumor tissue indicated intramural uterine leiomyoma.Patient history included hypothyroidism, pelvic floor relaxation, andparaplegia. Family history included benign hypertension, type IIdiabetes, and hyperlipidemia.

[0407] TABLE 7 Program Description Reference Parameter ThresholdABIFACTURA A program that removes vector sequences and AppliedBiosystems, Foster City, CA. masks ambiguous bases in nucleic acidsequences. ABI/ A Fast Data Finder useful in comparing and AppliedBiosystems, Foster City, CA; Mismatch <50% PARACEL annotating amino acidor nucleic acid sequences. Paracel Inc., Pasadena, CA. FDF ABI A programthat assembles nucleic acid sequences. Applied Biosystems, Foster City,CA. AutoAssembler BLAST A Basic Local Alignment Search Tool useful inAltschul, S. F. et al. (1990) J. Mol. Biol. ESTs: Probability sequencesimilarity search for amino acid and 215: 403-410; Altschul, S. F. etal. (1997) value = 1.0E−8 nucleic acid sequences. BLAST includes fiveNucleic Acids Res. 25: 3389-3402. or less functions: blastp, blastn,blastx, tblastn, and tblastx. Full Length sequences: Probability value =1.0E−10 or less FASTA A Pearson and Lipman algorithm that searches forPearson, W. R. and D. J. Lipman (1988) Proc. ESTs: fasta similaritybetween a query sequence and a group of Natl. Acad Sci. USA 85:2444-2448; E value = 1.06E−6 sequences of the same type. FASTA comprisesas Pearson, W. R. (1990) Methods Enzymol. Assembled ESTs: least fivefunctions: fasta, tfasta, fastx, tfastx, and 183: 63-98; and Smith, T.F. and fasta ldentity = 95% ssearch. M. S. Waterman (1981) or greaterand Adv. Appl. Math. 2: 482-489. Match length = 200 bases or greater,fastx E value = 1.0E−8 or less Full Length sequences: fastx score = 100or greater BLIMPS A BLocks IMProved Searcher that matches a Henikoff, S.and J. G. Henikoff (1991) Nucleic Probability sequence against those inBLOCKS, PRINTS, Acids Res. 19: 6565-6572; Henikoff, J. G. and value =1.0E−3 DOMO, PRODOM, and PFAM databases to search S. Henikoff (1996)Methods Enzymol. or less for gene families, sequence homology, and 266:88-105; and Attwood, T. K. et al. (1997) structural fingerprint regions.J. Chem. Inf. Comput. Sci. 37: 417-424. HMMER An algorithm for searchinga query sequence against Krogh, A. et al. (1994) J. Mol. Biol. PFAMhits: hidden Markov model (HMM)-based databases of 235: 1501-1531;Sonnhammer, E. L. L. et al. Probability protein family consensussequences, such as PFAM. (1988) Nucleic Acids Res. 26: 320-322; value =1.0E−3 Durbin, R. et al. (1998) Our World View, in a or less Nutshell,Cambridge Univ. Press, pp. 1-350. Signal peptide hits: Score = 0 orgreater ProfileScan An algorithm that searches for structural andsequence Gribskov, M. et al. (1988) CABIOS 4: 61-66; Normalized qualitymotifs in protein sequences that match sequence patterns Gribskov, M. etal. (1989) Methods Enzymol. score ≧ GCG- defined in Prosite. 183:146-159; Bairoch, A. et al. (1997) specified “HIGH” value Nucleic AcidsRes. 25: 217-221. for that particular Prosite motif. Generally, score =1.4-2.1. Phred A base-calling algorithm that examines automated Ewing,B. et al. (1998) Genome Res. sequencer traces with high sensitivity andprobability. 8: 175-185; Ewing, B. and P. Green (1998) Genome Res. 8:186-194. Phrap A Phils Revised Assembly Program including SWAT andSmith, T. F. and M. S. Waterman (1981) Adv. Score = 120 CrossMatch,programs based on efficient implementation Appl. Math. 2: 482-489;Smith, T. F. and or greater; of the Smith-Waterman algorithm, useful insearching M. S. Waterman (1981) J. Mol. Biol. 147: Match length = 56sequence homology and assembling DNA sequences. 195-197; and Green, P.,University or greater of Washington, Seattle, WA. Consed A graphicaltool for viewing and editing Phrap Gordon, D. et al. (1998) Genomeassemblies. Res. 8: 195-202. SPScan A weight matrix analysis programthat scans protein Nielson, H. et al. (1997) Protein Engineering Score =3.5 sequences for the presence of secretory signal peptides. 10: 1-6;Claverie, J. M. and S. Audic (1997) or greater CABIOS 12: 431-439. TMAPA program that uses weight matrices to delineate Persson, B. and P.Argos (1994) J. Mol. Biol. transmembrane segments on protein sequencesand 237: 182-192; Persson, B. and P. Argos (1996) determine orientation.Protein Sci. 5: 363-371. TMHMMER A program that uses a hidden Markovmodel (HMM) to Sonnhammer, E. L. et al. (1998) Proc. Sixth delineatetransmembrane segments on protein sequences Intl. Conf. on IntelligentSystems for and determine orientation. Mol. Biol., Glasgow et al., eds.,The Am. Assoc. for Artificial Intelligence Press, Menlo Park, CA, pp.175-182. Motifs A program that searches amino acid sequences forpatterns Bairoch, A. et al. (1997) Nucleic Acids that matched thosedefined in Prosite. Res. 25: 217-221; Wisconsin Package Program Manual,version 9, page M51-59, Genetics Computer Group, Madison, WI.

[0408]

1 18 1 996 PRT Homo sapiens misc_feature Incyte ID No 7472774CD1 1 MetLys Arg Ser Arg Pro Met His Pro Ile Cys Leu Pro Thr Gln 1 5 10 15 ThrThr Pro Arg Ala Ile Pro Ala Thr Ala Lys Leu Trp Pro Gly 20 25 30 Arg TrpSer Ser Glu Ser Glu Tyr Lys Phe Leu Ile Leu Pro Pro 35 40 45 Ser Trp ArgAla Ala Val Met Leu Leu Arg Gln Met His Ala Arg 50 55 60 Val Ser His SerLeu Pro Asp Pro Cys Gln Ala Glu Asp Ser Arg 65 70 75 Pro Ser Ala Thr CysAla Leu Lys Ala Pro Gln Thr Ser Trp Asp 80 85 90 Gly Leu Leu Arg Glu GlyLeu Ser Pro Cys His Leu Leu Thr Val 95 100 105 Arg Val Ile Arg Met LysAsn Val Arg Gln Ala Asp Met Gln Pro 110 115 120 Val Gly Ile Glu Leu AlaPro Cys Leu Gln Ala Pro Ser Val Pro 125 130 135 Glu Thr Asp Leu Lys GlyVal Val Gln Ala Arg Gly Gly Gly Ala 140 145 150 Ser Val Leu Glu Lys ProArg Glu Gly Phe Lys Arg Ala Glu Gln 155 160 165 Val Pro Val Ser Gln ThrAsp Cys Phe Val Ser Leu Trp Leu Pro 170 175 180 Thr Ala Ser Gln Lys LysLeu Arg Thr Arg Thr Ile Ser Asn Cys 185 190 195 Pro Asn Pro Glu Trp AsnGlu Ser Phe Asn Phe Gln Ile Gln Ser 200 205 210 Arg Val Lys Asn Val LeuGlu Leu Ser Val Cys Asp Glu Asp Thr 215 220 225 Val Thr Pro Asp Asp HisLeu Leu Thr Val Leu Tyr Asp Leu Thr 230 235 240 Lys Leu Cys Phe Arg LysLys Thr His Val Lys Phe Pro Leu Asn 245 250 255 Pro Gln Gly Met Glu GluLeu Glu Val Glu Phe Leu Leu Glu Glu 260 265 270 Ser Pro Ser Pro Pro GluThr Leu Val Thr Asn Gly Val Leu Val 275 280 285 Val Ile Ile Phe Leu GlySer Cys Ser Ser Arg Gly His Gly Trp 290 295 300 Leu Leu Leu Ser Gly GluGln Asp Gln Gly Arg Lys Gln Trp Ala 305 310 315 Gln Leu Gly Leu Cys ProIle Leu Thr Ser Ala Gly Val Arg Leu 320 325 330 Asn Glu Ala Ser Gln MetGly His Arg Gln His Trp Gly Thr Ser 335 340 345 Trp Gly Phe Cys Thr GluGly Gly Val Lys Asp Leu Leu Val Met 350 355 360 Val Asn Glu Ser Phe GluAsn Thr Gln Arg Val Arg Pro Cys Leu 365 370 375 Glu Pro Cys Cys Pro ThrSer Ala Cys Phe Gln Thr Ala Ala Cys 380 385 390 Phe His Tyr Pro Lys TyrPhe Gln Ser Gln Val His Val Glu Val 395 400 405 Pro Lys Ser His Trp SerCys Gly Leu Cys Cys Arg Ser Arg Lys 410 415 420 Lys Gly Pro Ile Ser GlnPro Leu Asp Cys Leu Ser Asp Gly Gln 425 430 435 Val Met Thr Leu Pro ValGly Glu Ser Tyr Glu Leu His Met Lys 440 445 450 Ser Thr Pro Cys Pro GluThr Leu Asp Val Arg Leu Gly Phe Ser 455 460 465 Leu Cys Pro Ala Glu LeuGlu Phe Leu Gln Lys Arg Lys Val Val 470 475 480 Val Ala Lys Ala Leu LysGln Val Leu Gln Leu Glu Glu Asp Leu 485 490 495 Gln Glu Asp Glu Val ProLeu Ile Ala Ile Met Ala Thr Gly Gly 500 505 510 Gly Thr Arg Ser Met ThrSer Met Tyr Gly His Leu Leu Gly Leu 515 520 525 Gln Lys Leu Asn Leu LeuAsp Cys Ala Ser Tyr Ile Thr Gly Leu 530 535 540 Ser Gly Ala Thr Trp ThrMet Ala Thr Leu Tyr Arg Asp Pro Asp 545 550 555 Trp Ser Ser Lys Asn LeuGlu Pro Ala Ile Phe Glu Ala Arg Arg 560 565 570 His Val Val Lys Asp LysLeu Pro Ser Leu Phe Pro Asp Gln Leu 575 580 585 Arg Lys Phe Gln Glu GluLeu Arg Gln Arg Ser Gln Glu Gly Tyr 590 595 600 Arg Val Thr Phe Thr AspPhe Trp Gly Leu Leu Ile Glu Thr Cys 605 610 615 Leu Gly Asp Glu Arg AsnGlu Cys Lys Leu Ser Asp Gln Arg Ala 620 625 630 Ala Leu Ser Cys Gly GlnAsn Pro Leu Pro Ile Tyr Leu Thr Ile 635 640 645 Asn Val Lys Asp Asp ValSer Asn Gln Asp Val Arg Trp Phe Glu 650 655 660 Phe Ser Pro Tyr Glu ValGly Leu Gln Lys Tyr Gly Ala Phe Ile 665 670 675 Pro Ser Glu Leu Phe GlySer Glu Phe Phe Met Gly Arg Leu Val 680 685 690 Lys Arg Ile Pro Glu SerArg Ile Cys Tyr Met Leu Gly Leu Trp 695 700 705 Ser Ser Ile Phe Ser LeuAsn Leu Leu Asp Ala Trp Asn Leu Ser 710 715 720 His Thr Ser Glu Glu PhePhe His Arg Trp Thr Arg Glu Lys Val 725 730 735 Gln Asp Ile Glu Asp GluPro Ile Leu Pro Glu Ile Pro Lys Cys 740 745 750 Asp Ala Asn Ile Leu GluThr Thr Val Val Ile Pro Gly Ser Trp 755 760 765 Leu Ser Asn Ser Phe ArgGlu Ile Leu Thr His Arg Ser Phe Val 770 775 780 Ser Glu Phe His Asn PheLeu Ser Gly Leu Gln Leu His Thr Asn 785 790 795 Tyr Leu Gln Asn Gly GlnPhe Ser Arg Trp Lys Asp Thr Val Leu 800 805 810 Asp Gly Phe Pro Asn GlnLeu Thr Glu Ser Ala Asn His Leu Cys 815 820 825 Leu Leu Asp Thr Ala PhePhe Val Asn Ser Ser Tyr Pro Pro Leu 830 835 840 Leu Arg Pro Glu Arg LysAla Asp Leu Ile Ile His Leu Asn Tyr 845 850 855 Cys Ala Gly Ser Gln ThrLys Pro Leu Lys Gln Thr Cys Glu Tyr 860 865 870 Cys Thr Val Gln Asn IlePro Phe Pro Lys Tyr Glu Leu Pro Asp 875 880 885 Glu Asn Glu Asn Leu LysGlu Cys Tyr Leu Met Glu Asn Pro Gln 890 895 900 Glu Pro Asp Ala Pro IleVal Thr Phe Phe Pro Leu Ile Asn Asp 905 910 915 Thr Phe Arg Lys Tyr LysAla Pro Gly Val Glu Arg Ser Pro Glu 920 925 930 Glu Leu Glu Gln Gly GlnVal Asp Ile Tyr Gly Pro Lys Thr Pro 935 940 945 Tyr Ala Thr Lys Glu LeuThr Tyr Thr Glu Ala Thr Phe Asp Lys 950 955 960 Leu Val Lys Leu Ser GluTyr Asn Ile Leu Asn Asn Lys Asp Thr 965 970 975 Leu Leu Gln Ala Leu ArgLeu Ala Val Glu Lys Lys Lys Arg Leu 980 985 990 Lys Gly Gln Cys Pro Ser995 2 372 PRT Homo sapiens misc_feature Incyte ID No 2884821CD1 2 MetSer Leu Arg Cys Gly Asp Ala Ala Arg Thr Leu Gly Pro Arg 1 5 10 15 ValPhe Gly Arg Tyr Phe Cys Ser Pro Val Arg Pro Leu Ser Ser 20 25 30 Leu ProAsp Lys Lys Lys Glu Leu Leu Gln Asn Gly Pro Asp Leu 35 40 45 Gln Asp PheVal Ser Gly Asp Leu Ala Asp Arg Ser Thr Trp Asp 50 55 60 Glu Tyr Lys GlyAsn Leu Lys Arg Gln Lys Gly Glu Arg Leu Arg 65 70 75 Leu Pro Pro Trp LeuLys Thr Glu Ile Pro Met Gly Lys Asn Tyr 80 85 90 Asn Lys Leu Lys Asn ThrLeu Arg Asn Leu Asn Leu His Thr Val 95 100 105 Cys Glu Glu Ala Arg CysPro Asn Ile Gly Glu Cys Trp Gly Gly 110 115 120 Gly Glu Tyr Ala Thr AlaThr Ala Thr Ile Met Leu Met Gly Asp 125 130 135 Thr Cys Thr Arg Gly CysArg Phe Cys Ser Val Lys Thr Ala Arg 140 145 150 Asn Pro Pro Pro Leu AspAla Ser Glu Pro Tyr Asn Thr Ala Lys 155 160 165 Ala Ile Ala Glu Trp GlyLeu Asp Tyr Val Val Leu Thr Ser Val 170 175 180 Asp Arg Asp Asp Met ProAsp Gly Gly Ala Glu His Ile Ala Lys 185 190 195 Thr Val Ser Tyr Leu LysGlu Arg Asn Pro Lys Ile Leu Val Glu 200 205 210 Cys Leu Thr Pro Asp PheArg Gly Asp Leu Lys Ala Ile Glu Lys 215 220 225 Val Ala Leu Ser Gly LeuAsp Val Tyr Ala His Asn Val Glu Thr 230 235 240 Val Pro Glu Leu Gln SerLys Val Arg Asp Pro Arg Ala Asn Phe 245 250 255 Asp Gln Ser Leu Arg ValLeu Lys His Ala Lys Lys Val Gln Pro 260 265 270 Asp Val Ile Ser Lys ThrSer Ile Met Leu Gly Leu Gly Glu Asn 275 280 285 Asp Glu Gln Val Tyr AlaThr Met Lys Ala Leu Arg Glu Ala Asp 290 295 300 Val Asp Cys Leu Thr LeuGly Gln Tyr Met Gln Pro Thr Arg Arg 305 310 315 His Leu Lys Val Glu GluTyr Ile Thr Pro Glu Lys Phe Lys Tyr 320 325 330 Trp Glu Lys Val Gly AsnGlu Leu Gly Phe His Tyr Thr Ala Ser 335 340 345 Gly Pro Leu Val Arg SerSer Tyr Lys Ala Gly Glu Phe Phe Leu 350 355 360 Lys Asn Leu Val Ala LysArg Lys Thr Lys Asp Leu 365 370 3 649 PRT Homo sapiens misc_featureIncyte ID No 72852842CD1 3 Met Glu Met Arg Trp Phe Leu Ser Lys Ile GlnAsp Asp Phe Arg 1 5 10 15 Gly Gly Lys Ile Asn Leu Glu Lys Thr Gln ArgLeu Leu Glu Lys 20 25 30 Leu Asp Ile Arg Cys Ser Tyr Ile His Val Lys GlnIle Phe Lys 35 40 45 Asp Asn Asp Arg Leu Lys Gln Gly Arg Ile Thr Ile GluGlu Phe 50 55 60 Arg Ala Ile Tyr Arg Ile Ile Thr His Arg Glu Glu Ile IleGlu 65 70 75 Ile Phe Asn Thr Tyr Ser Glu Asn Arg Lys Ile Leu Leu Ala Ser80 85 90 Asn Leu Ala Gln Phe Leu Thr Gln Glu Gln Tyr Ala Ala Glu Met 95100 105 Ser Lys Ala Ile Ala Phe Glu Ile Ile Gln Lys Tyr Glu Pro Ile 110115 120 Glu Glu Val Arg Lys Ala His Gln Met Ser Leu Glu Gly Phe Thr 125130 135 Arg Tyr Met Asp Ser Arg Glu Cys Leu Leu Phe Lys Asn Glu Cys 140145 150 Arg Lys Val Tyr Gln Asp Met Thr His Pro Leu Asn Asp Tyr Phe 155160 165 Ile Ser Ser Ser His Asn Thr Tyr Leu Val Ser Asp Gln Leu Leu 170175 180 Gly Pro Ser Asp Leu Trp Gly Tyr Val Ser Ala Leu Val Lys Gly 185190 195 Cys Arg Cys Leu Glu Ile Asp Cys Trp Asp Gly Ala Gln Asn Glu 200205 210 Pro Val Val Tyr His Gly Tyr Thr Leu Thr Ser Lys Leu Leu Phe 215220 225 Lys Thr Val Ile Gln Ala Ile His Lys Tyr Ala Phe Met Thr Ser 230235 240 Asp Tyr Pro Val Val Leu Ser Leu Glu Asn His Cys Ser Thr Ala 245250 255 Gln Gln Glu Val Met Ala Asp Asn Leu Gln Ala Thr Phe Gly Glu 260265 270 Ser Leu Leu Ser Asp Met Leu Asp Asp Phe Pro Asp Thr Leu Pro 275280 285 Ser Pro Glu Ala Leu Lys Phe Lys Ile Leu Val Lys Asn Lys Lys 290295 300 Ile Gly Thr Leu Lys Glu Thr His Glu Arg Lys Gly Ser Asp Lys 305310 315 Arg Gly Lys Val Glu Glu Trp Glu Glu Glu Val Ala Asp Gly Glu 320325 330 Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Asp 335340 345 Lys Phe Lys Glu Ser Glu Val Leu Glu Ser Val Leu Gly Asp Asn 350355 360 Gln Asp Lys Glu Thr Gly Val Lys Lys Leu Pro Gly Val Met Leu 365370 375 Phe Lys Lys Lys Lys Thr Arg Lys Leu Lys Ile Ala Leu Ala Leu 380385 390 Ser Asp Leu Val Ile Tyr Thr Lys Ala Glu Lys Phe Lys Ser Phe 395400 405 Gln His Ser Arg Leu Tyr Gln Gln Phe Asn Glu Asn Asn Ser Ile 410415 420 Gly Glu Thr Gln Ala Arg Lys Leu Ser Lys Leu Arg Val His Glu 425430 435 Phe Ile Phe His Thr Arg Lys Phe Ile Thr Arg Ile Tyr Pro Lys 440445 450 Ala Thr Arg Ala Asp Ser Ser Asn Phe Asn Pro Gln Glu Phe Trp 455460 465 Asn Ile Gly Cys Gln Met Val Ala Leu Asn Phe Gln Thr Pro Gly 470475 480 Leu Pro Met Asp Leu Gln Asn Gly Lys Phe Leu Asp Asn Gly Gly 485490 495 Ser Gly Tyr Ile Leu Lys Pro His Phe Leu Arg Glu Ser Lys Ser 500505 510 Tyr Phe Asn Pro Ser Asn Ile Lys Glu Gly Met Pro Ile Thr Leu 515520 525 Thr Ile Arg Leu Ile Ser Gly Ile Gln Leu Pro Leu Thr His Ser 530535 540 Ser Ser Asn Lys Gly Asp Ser Leu Val Ile Ile Glu Val Phe Gly 545550 555 Val Pro Asn Asp Gln Met Lys Gln Gln Thr Arg Val Ile Lys Lys 560565 570 Asn Ala Phe Ser Pro Arg Trp Asn Glu Thr Phe Thr Phe Ile Ile 575580 585 His Val Pro Glu Leu Ala Leu Ile Arg Phe Val Val Glu Gly Gln 590595 600 Gly Leu Ile Ala Gly Asn Glu Phe Leu Gly Gln Tyr Thr Leu Pro 605610 615 Leu Leu Cys Met Asn Lys Gly Tyr Arg Arg Ile Pro Leu Phe Ser 620625 630 Arg Met Gly Glu Ser Leu Glu Pro Ala Ser Leu Phe Val Tyr Val 635640 645 Trp Tyr Val Arg 4 2020 PRT Homo sapiens misc_feature Incyte IDNo 7484271CD1 4 Met Ser Gly Gly Leu Val Pro Ile Tyr Val Ile Ala Gly ValVal 1 5 10 15 Thr Arg Lys Gly Arg Arg Gly Trp Asp Ile Met Met Gln LeuThr 20 25 30 Leu Asn Thr Leu Phe Pro Val Val Ser Thr Pro Ala Ile Thr Tyr35 40 45 Ile Val Thr Val Phe Thr Gly Asp Val Arg Gly Ala Gly Thr Lys 5055 60 Ser Lys Ile Tyr Leu Val Met Tyr Gly Ala Arg Gly Asn Lys Asn 65 7075 Ser Gly Lys Ile Phe Leu Glu Gly Gly Val Phe Asp Arg Gly Arg 80 85 90Thr Asp Ile Phe His Ile Glu Leu Ala Val Leu Leu Ser Pro Leu 95 100 105Ser Arg Val Ser Val Gly His Gly Asn Val Gly Val Asn Arg Gly 110 115 120Trp Phe Cys Glu Lys Val Val Ile Leu Cys Pro Phe Thr Gly Ile 125 130 135Gln Gln Thr Phe Pro Cys Ser Asn Trp Leu Asp Glu Lys Lys Ala 140 145 150Asp Gly Leu Ile Glu Arg Gln Leu Tyr Glu Met Val Ser Leu Arg 155 160 165Lys Lys Arg Leu Lys Lys Phe Pro Trp Ser Leu Trp Val Trp Thr 170 175 180Thr Asp Leu Lys Lys Ala Gly Thr Asn Ser Pro Ile Phe Ile Gln 185 190 195Ile Tyr Gly Gln Lys Gly Arg Thr Asp Glu Ile Leu Leu Asn Pro 200 205 210Asn Asn Lys Trp Phe Lys Pro Gly Ile Ile Glu Lys Phe Arg Ile 215 220 225Glu Leu Pro Asp Leu Gly Arg Phe Tyr Lys Ile Arg Val Trp His 230 235 240Asp Lys Arg Ser Ser Gly Ser Gly Trp His Leu Glu Arg Met Thr 245 250 255Leu Met Asn Thr Leu Asn Lys Asp Lys Tyr Asn Phe Asn Cys Asn 260 265 270Arg Trp Leu Asp Ala Asn Glu Asp Asp Asn Glu Ile Val Arg Glu 275 280 285Met Thr Ala Glu Gly Pro Thr Val Arg Arg Ile Met Gly Met Ala 290 295 300Arg Tyr His Val Thr Val Cys Thr Gly Glu Leu Glu Gly Ala Gly 305 310 315Thr Asp Ala Asn Val Tyr Leu Cys Leu Phe Gly Asp Val Gly Asp 320 325 330Thr Gly Glu Arg Leu Leu Tyr Asn Cys Arg Asn Asn Thr Asp Leu 335 340 345Phe Glu Lys Gly Asn Ala Asp Glu Phe Thr Ile Glu Ser Val Thr 350 355 360Met Arg Asn Val Arg Arg Val Arg Ile Arg His Asp Gly Lys Gly 365 370 375Ser Gly Ser Gly Trp Tyr Leu Asp Arg Val Leu Val Arg Glu Glu 380 385 390Gly Gln Pro Glu Ser Asp Asn Val Glu Phe Pro Cys Leu Arg Trp 395 400 405Leu Asp Lys Asp Lys Asp Asp Gly Gln Leu Val Arg Glu Leu Leu 410 415 420Pro Ser Asp Ser Ser Ala Thr Leu Lys Asn Phe Arg Tyr His Ile 425 430 435Ser Leu Lys Thr Gly Asp Val Ser Gly Ala Ser Thr Asp Ser Arg 440 445 450Val Tyr Ile Lys Leu Tyr Gly Asp Lys Ser Asp Thr Ile Lys Gln 455 460 465Val Leu Leu Val Ser Asp Asn Asn Leu Lys Asp Tyr Phe Glu Arg 470 475 480Gly Arg Val Asp Glu Phe Thr Leu Glu Thr Leu Asn Ile Gly Asn 485 490 495Ile Asn Arg Leu Val Ile Gly His Asp Ser Thr Gly Met His Ala 500 505 510Ser Trp Phe Leu Gly Ser Val Gln Ile Arg Val Pro Arg Gln Gly 515 520 525Lys Gln Tyr Thr Phe Pro Ala Asn Arg Trp Leu Asp Lys Asn Gln 530 535 540Ala Asp Gly Arg Leu Glu Val Glu Leu Tyr Pro Ser Glu Val Val 545 550 555Glu Ile Gln Lys Leu Val His Tyr Glu Val Glu Ile Trp Thr Gly 560 565 570Asp Val Gly Gly Ala Gly Thr Ser Ala Arg Val Tyr Met Gln Ile 575 580 585Tyr Gly Glu Lys Gly Lys Thr Glu Val Leu Phe Leu Ser Ser Arg 590 595 600Ser Lys Val Phe Glu Arg Ala Ser Lys Asp Thr Phe Gln Leu Glu 605 610 615Ala Ala Asp Val Gly Glu Val Tyr Lys Leu Arg Leu Gly His Thr 620 625 630Gly Glu Gly Phe Gly Pro Ser Trp Phe Val Asp Thr Val Trp Leu 635 640 645Arg His Leu Val Val Arg Glu Val Asp Leu Thr Pro Glu Glu Glu 650 655 660Ala Arg Lys Lys Lys Glu Lys Asp Lys Leu Arg Gln Leu Leu Lys 665 670 675Lys Glu Arg Leu Lys Ala Lys Leu Gln Arg Lys Lys Lys Lys Arg 680 685 690Lys Gly Ser Asp Glu Glu Asp Glu Gly Glu Glu Glu Glu Ser Ser 695 700 705Ser Ser Glu Glu Ser Ser Ser Glu Glu Glu Glu Met Glu Glu Glu 710 715 720Glu Glu Glu Glu Glu Phe Gly Pro Gly Met Gln Glu Val Ile Glu 725 730 735Gln His Lys Phe Glu Ala His Arg Trp Leu Ala Arg Gly Lys Glu 740 745 750Asp Asn Glu Leu Val Val Glu Leu Val Pro Ala Gly Lys Pro Gly 755 760 765Pro Glu Arg Asn Thr Tyr Glu Val Gln Val Val Thr Gly Asn Val 770 775 780Pro Lys Ala Gly Thr Asp Ala Asn Val Tyr Leu Thr Ile Tyr Gly 785 790 795Glu Glu Tyr Gly Asp Thr Gly Glu Arg Pro Leu Lys Lys Ser Asp 800 805 810Lys Ser Asn Lys Phe Glu Gln Gly Gln Thr Asp Thr Phe Thr Ile 815 820 825Tyr Ala Ile Asp Leu Gly Ala Leu Thr Lys Ile Arg Ile Arg His 830 835 840Asp Asn Thr Gly Asn Arg Ala Gly Trp Phe Leu Asp Arg Ile Asp 845 850 855Ile Thr Asp Met Asn Asn Glu Ile Thr Tyr Tyr Phe Pro Cys Gln 860 865 870Arg Trp Leu Ala Val Glu Glu Asp Asp Gly Gln Leu Ser Arg Glu 875 880 885Leu Leu Pro Val Asp Glu Ser Tyr Val Leu Pro Gln Ser Glu Glu 890 895 900Gly Gly Gly Gly Gly Asp Asn Asn Pro Leu Asp Asn Leu Ala Leu 905 910 915Glu Gln Lys Asp Lys Ser Thr Thr Phe Ser Val Thr Ile Lys Thr 920 925 930Gly Val Lys Lys Asn Ala Gly Thr Asp Ala Asn Val Phe Ile Thr 935 940 945Leu Phe Gly Thr Gln Asp Asp Thr Gly Met Thr Leu Leu Lys Ser 950 955 960Ser Lys Thr Asn Ser Asp Lys Phe Glu Arg Asp Ser Ile Glu Ile 965 970 975Phe Thr Val Glu Thr Leu Asp Leu Gly Asp Leu Trp Lys Val Arg 980 985 990Leu Gly His Asp Asn Thr Gly Lys Ala Pro Gly Trp Phe Val Asp 995 10001005 Trp Val Glu Val Asp Ala Pro Ser Leu Gly Lys Cys Met Thr Phe 10101015 1020 Pro Cys Gly Arg Trp Leu Ala Lys Asn Glu Asp Asp Gly Ser Ile1025 1030 1035 Ile Arg Asp Leu Phe His Ala Glu Leu Gln Thr Arg Leu TyrThr 1040 1045 1050 Pro Phe Val Pro Tyr Glu Ile Thr Leu Tyr Thr Ser AspVal Phe 1055 1060 1065 Ala Ala Gly Thr Asp Ala Asn Ile Phe Ile Ile IleTyr Gly Cys 1070 1075 1080 Asp Ala Val Cys Thr Gln Gln Lys Tyr Leu CysThr Asn Lys Arg 1085 1090 1095 Glu Gln Lys Gln Phe Phe Glu Arg Lys SerAla Ser Arg Phe Ile 1100 1105 1110 Val Glu Leu Glu Asp Val Gly Glu IleIle Glu Lys Ile Arg Ile 1115 1120 1125 Gly His Asn Asn Thr Gly Met AsnPro Gly Trp His Cys Ser His 1130 1135 1140 Val Asp Ile Arg Arg Leu LeuPro Asp Lys Asp Gly Ala Glu Thr 1145 1150 1155 Leu Thr Phe Pro Cys AspArg Trp Leu Ala Thr Ser Glu Asp Asp 1160 1165 1170 Lys Lys Thr Ile ArgGlu Leu Val Pro Tyr Asp Ile Phe Thr Glu 1175 1180 1185 Lys Tyr Met LysAsp Gly Ser Leu Arg Gln Val Tyr Lys Glu Val 1190 1195 1200 Glu Glu ProLeu Asp Ile Val Leu Tyr Ser Val Gln Ile Phe Thr 1205 1210 1215 Gly AsnIle Pro Gly Ala Gly Thr Asp Ala Lys Val Tyr Ile Thr 1220 1225 1230 IleTyr Gly Asp Leu Gly Asp Thr Gly Glu Arg Tyr Leu Gly Lys 1235 1240 1245Ser Glu Asn Arg Thr Asn Lys Phe Glu Arg Gly Thr Ala Asp Thr 1250 12551260 Phe Ile Ile Glu Ala Ala Asp Leu Gly Val Ile Tyr Lys Ile Lys 12651270 1275 Leu Arg His Asp Asn Ser Lys Trp Cys Ala Asp Trp Tyr Val Glu1280 1285 1290 Lys Val Glu Ile Trp Asn Asp Thr Asn Glu Asp Glu Phe LeuPhe 1295 1300 1305 Leu Cys Gly Arg Trp Leu Ser Leu Lys Lys Glu Asp GlyArg Leu 1310 1315 1320 Glu Arg Leu Phe Tyr Glu Lys Glu Tyr Thr Gly AspArg Ser Ser 1325 1330 1335 Asn Cys Ser Ser Pro Ala Asp Phe Trp Glu IleAla Leu Ser Ser 1340 1345 1350 Lys Met Ala Asp Val Asp Ile Ser Thr ValThr Gly Pro Met Ala 1355 1360 1365 Asp Tyr Val Gln Glu Gly Pro Ile IlePro Tyr Tyr Val Ser Val 1370 1375 1380 Thr Thr Gly Lys His Lys Asp AlaAla Thr Asp Ser Arg Ala Phe 1385 1390 1395 Ile Phe Leu Ile Gly Glu AspAsp Glu Arg Ser Lys Arg Ile Trp 1400 1405 1410 Leu Asp Tyr Pro Arg GlyLys Arg Gly Phe Ser Arg Gly Ser Val 1415 1420 1425 Glu Glu Phe Tyr ValAla Gly Leu Asp Val Gly Ile Ile Lys Lys 1430 1435 1440 Ile Glu Leu GlyHis Asp Gly Ala Ser Pro Glu Ser Cys Trp Leu 1445 1450 1455 Val Glu GluLeu Cys Leu Ala Val Pro Thr Gln Gly Thr Lys Tyr 1460 1465 1470 Met LeuAsn Cys Asn Cys Trp Leu Ala Lys Asp Arg Gly Asp Gly 1475 1480 1485 IleThr Ser Arg Val Phe Asp Leu Leu Asp Ala Met Val Val Asn 1490 1495 1500Ile Gly Val Lys Val Leu Tyr Glu Met Thr Val Trp Thr Gly Asp 1505 15101515 Val Val Gly Gly Gly Thr Asp Ser Asn Ile Phe Met Thr Leu Tyr 15201525 1530 Gly Ile Asn Gly Ser Thr Glu Glu Met Gln Leu Asp Lys Lys Lys1535 1540 1545 Ala Arg Phe Glu Arg Glu Gln Asn Asp Thr Phe Ile Met GluIle 1550 1555 1560 Leu Asp Ile Ala Pro Phe Thr Lys Met Arg Ile Arg IleAsp Gly 1565 1570 1575 Leu Gly Ser Arg Pro Glu Trp Phe Leu Glu Arg IleLeu Leu Lys 1580 1585 1590 Asn Met Asn Thr Gly Asp Leu Thr Met Phe TyrTyr Gly Asp Trp 1595 1600 1605 Leu Ser Gln Arg Lys Gly Lys Lys Thr LeuVal Cys Glu Met Cys 1610 1615 1620 Ala Val Ile Asp Glu Glu Glu Met MetGlu Trp Thr Ser Tyr Thr 1625 1630 1635 Val Ala Val Lys Thr Ser Asp IleLeu Gly Ala Gly Thr Asp Ala 1640 1645 1650 Asn Val Phe Ile Ile Ile PheGly Glu Asn Gly Asp Ser Gly Thr 1655 1660 1665 Leu Ala Leu Lys Gln SerAla Asn Trp Asn Lys Phe Glu Arg Asn 1670 1675 1680 Asn Thr Asp Thr PheAsn Phe Pro Asp Met Leu Ser Leu Gly His 1685 1690 1695 Leu Cys Lys LeuArg Val Trp His Asp Asn Lys Gly Ile Phe Pro 1700 1705 1710 Gly Trp HisLeu Ser Tyr Val Asp Val Lys Asp Asn Ser Arg Asp 1715 1720 1725 Glu ThrPhe His Phe Gln Cys Asp Cys Trp Leu Ser Lys Ser Glu 1730 1735 1740 GlyAsp Gly Gln Thr Val Arg Asp Phe Ala Cys Ala Asn Asn Lys 1745 1750 1755Ile Cys Asp Glu Leu Glu Glu Thr Thr Tyr Glu Ile Val Ile Glu 1760 17651770 Thr Gly Asn Gly Gly Glu Thr Arg Glu Asn Val Trp Leu Ile Leu 17751780 1785 Glu Gly Arg Lys Asn Arg Ser Lys Glu Phe Leu Met Glu Asn Ser1790 1795 1800 Ser Arg Gln Arg Ala Phe Arg Lys Gly Thr Thr Asp Thr PheGlu 1805 1810 1815 Phe Asp Ser Ile Tyr Leu Gly Asp Ile Ala Ser Leu CysVal Gly 1820 1825 1830 His Leu Ala Arg Glu Asp Arg Phe Ile Pro Lys ArgGlu Leu Ala 1835 1840 1845 Trp His Val Lys Thr Ile Thr Ile Thr Glu MetGlu Tyr Gly Asn 1850 1855 1860 Val Tyr Phe Phe Asn Cys Asp Cys Leu IlePro Leu Lys Arg Lys 1865 1870 1875 Arg Lys Tyr Phe Lys Val Phe Glu ValThr Lys Thr Thr Glu Ser 1880 1885 1890 Phe Ala Ser Lys Val Gln Ser LeuVal Pro Val Lys Tyr Glu Val 1895 1900 1905 Ile Val Thr Thr Gly Tyr GluPro Gly Ala Gly Thr Asp Ala Asn 1910 1915 1920 Val Phe Val Thr Ile PheGly Ala Asn Gly Asp Thr Gly Lys Arg 1925 1930 1935 Glu Leu Lys Gln LysMet Arg Asn Leu Phe Glu Arg Gly Ser Thr 1940 1945 1950 Asp Arg Phe PheLeu Glu Thr Leu Glu Leu Gly Glu Leu Arg Lys 1955 1960 1965 Val Arg LeuGlu His Asp Ser Ser Gly Tyr Cys Ser Gly Trp Leu 1970 1975 1980 Val GluLys Val Glu Val Thr Asn Thr Ser Thr Gly Val Ala Thr 1985 1990 1995 IlePhe Asn Cys Gly Arg Trp Leu Asp Lys Lys Arg Gly Asp Gly 2000 2005 2010Leu Thr Trp Arg Asp Leu Phe Pro Ser Val 2015 2020 5 415 PRT Homo sapiensmisc_feature Incyte ID No 7474074CD1 5 Met Met Tyr Thr Arg Asn Asn LeuAsn Cys Ala Glu Pro Leu Phe 1 5 10 15 Glu Gln Asn Asn Ser Leu Asn ValAsn Phe Asn Thr Gln Lys Lys 20 25 30 Thr Val Trp Leu Ile His Gly Tyr ArgPro Val Gly Ser Ile Pro 35 40 45 Leu Trp Leu Gln Asn Phe Val Arg Ile LeuLeu Asn Glu Glu Asp 50 55 60 Met Asn Val Ile Val Val Asp Trp Ser Arg GlyAla Thr Thr Phe 65 70 75 Ile Tyr Asn Arg Ala Val Lys Asn Thr Arg Lys ValAla Val Ser 80 85 90 Leu Ser Val His Ile Lys Asn Leu Leu Lys His Gly AlaSer Leu 95 100 105 Asp Asn Phe His Phe Ile Gly Val Ser Leu Gly Ala HisIle Ser 110 115 120 Gly Phe Val Gly Lys Ile Phe His Gly Gln Leu Gly ArgIle Thr 125 130 135 Gly Leu Asp Pro Ala Gly Pro Arg Phe Ser Arg Lys ProPro Tyr 140 145 150 Ser Arg Leu Asp Tyr Thr Asp Ala Lys Phe Val Asp ValIle His 155 160 165 Ser Asp Ser Asn Gly Leu Gly Ile Gln Glu Pro Leu GlyHis Ile 170 175 180 Asp Phe Tyr Pro Asn Gly Gly Asn Lys Gln Pro Gly CysPro Lys 185 190 195 Ser Ile Phe Ser Gly Ile Gln Phe Ile Lys Cys Asn HisGln Arg 200 205 210 Ala Val His Leu Phe Met Ala Ser Leu Glu Thr Asn CysAsn Phe 215 220 225 Ile Ser Phe Pro Cys Arg Ser Tyr Lys Asp Tyr Lys ThrSer Leu 230 235 240 Cys Val Asp Cys Asp Cys Phe Lys Glu Lys Ser Cys ProArg Leu 245 250 255 Gly Tyr Gln Ala Lys Leu Phe Lys Gly Val Leu Lys GluArg Met 260 265 270 Glu Gly Arg Pro Leu Arg Thr Thr Val Phe Leu Asp ThrSer Gly 275 280 285 Thr Tyr Pro Phe Cys Thr Tyr Tyr Phe Val Leu Ser IleIle Val 290 295 300 Pro Asp Lys Thr Met Met Asp Gly Ser Phe Ser Phe LysLeu Leu 305 310 315 Asn Gln Leu Glu Met Ile Glu Glu Pro Arg Leu Tyr GluLys Asn 320 325 330 Lys Pro Phe Tyr Lys Leu Gln Glu Val Lys Ile Leu AlaGln Phe 335 340 345 Tyr Asn Asp Phe Val Asn Ile Ser Ser Ile Gly Leu ThrTyr Phe 350 355 360 Gln Ser Ser Asn Leu Gln Cys Ser Thr Cys Thr Tyr LysIle Gln 365 370 375 Ser Leu Met Leu Lys Ser Leu Thr Tyr Pro Lys Arg ProPro Leu 380 385 390 Cys Arg Tyr Asn Ile Val Leu Lys Glu Arg Glu Glu ValPhe Leu 395 400 405 Asn Pro Asn Thr Cys Thr Pro Lys Asn Thr 410 415 61152 PRT Homo sapiens misc_feature Incyte ID No 72024970CD1 6 Met AlaLeu Pro Arg Gln Pro Asp Gln Gly Asn Gly Gly Leu Ala 1 5 10 15 Gly GlyGly Thr Pro Leu Val Gly Gly Ser Val Val Leu Ser Ser 20 25 30 Glu Trp GlnLeu Gly Pro Leu Val Glu Arg Cys Met Gly Ala Met 35 40 45 Gln Glu Gly MetGln Met Val Lys Leu Arg Gly Gly Ser Lys Gly 50 55 60 Leu Val Arg Phe TyrTyr Leu Asp Glu His Arg Ser Cys Ile Arg 65 70 75 Trp Arg Pro Ser Arg LysAsn Glu Lys Ala Lys Ile Ser Ile Asp 80 85 90 Ser Ile Gln Glu Val Ser GluGly Arg Gln Ser Glu Val Phe Gln 95 100 105 Arg Tyr Pro Asp Gly Ser PheAsp Pro Asn Cys Cys Phe Ser Ile 110 115 120 Tyr His Gly Ser His Arg GluSer Leu Asp Leu Val Ser Thr Ser 125 130 135 Ser Glu Val Ala Arg Thr TrpVal Thr Gly Leu Arg Tyr Leu Met 140 145 150 Ala Gly Ile Ser Asp Glu AspSer Leu Ala Arg Arg Gln Arg Thr 155 160 165 Arg Asp Gln Trp Leu Lys GlnThr Phe Asp Glu Ala Asp Lys Asn 170 175 180 Gly Asp Gly Ser Leu Ser IleGly Glu Val Leu Gln Leu Leu His 185 190 195 Lys Leu Asn Val Asn Leu ProArg Gln Arg Val Lys Gln Met Phe 200 205 210 Arg Glu Ala Asp Thr Asp AspHis Gln Gly Thr Leu Gly Phe Glu 215 220 225 Glu Phe Cys Ala Phe Tyr LysMet Met Ser Thr Arg Arg Asp Leu 230 235 240 Tyr Leu Leu Met Leu Thr TyrSer Asn His Lys Asp His Leu Asp 245 250 255 Ala Ala Ser Leu Gln Arg PheLeu Gln Val Glu Gln Lys Met Ala 260 265 270 Gly Val Thr Leu Glu Ser CysGln Asp Ile Ile Glu Gln Phe Glu 275 280 285 Pro Cys Pro Glu Asn Lys SerLys Gly Leu Leu Gly Ile Asp Gly 290 295 300 Phe Thr Asn Tyr Thr Arg SerPro Ala Gly Asp Ile Phe Asn Pro 305 310 315 Glu His His His Val His GlnAsp Met Thr Gln Pro Leu Ser His 320 325 330 Tyr Phe Ile Thr Ser Ser HisAsn Thr Tyr Leu Val Gly Asp Gln 335 340 345 Leu Met Ser Gln Ser Arg ValAsp Met Tyr Ala Trp Val Leu Gln 350 355 360 Ala Gly Cys Arg Cys Val GluVal Asp Cys Trp Asp Gly Pro Asp 365 370 375 Gly Glu Pro Ile Val His HisGly Tyr Thr Leu Thr Ser Lys Ile 380 385 390 Leu Phe Lys Asp Val Ile GluThr Ile Asn Lys Tyr Ala Phe Ile 395 400 405 Lys Asn Glu Tyr Pro Val IleLeu Ser Ile Glu Asn His Cys Ser 410 415 420 Val Ile Gln Gln Lys Lys MetAla Gln Tyr Leu Thr Asp Ile Leu 425 430 435 Gly Asp Lys Leu Asp Leu SerSer Val Ser Ser Glu Asp Ala Thr 440 445 450 Thr Leu Pro Ser Pro Gln MetLeu Lys Gly Lys Ile Leu Val Lys 455 460 465 Gly Lys Lys Leu Pro Ala AsnIle Ser Glu Asp Ala Glu Glu Gly 470 475 480 Glu Val Ser Asp Glu Asp SerAla Asp Glu Ile Asp Asp Asp Cys 485 490 495 Lys Leu Leu Asn Gly Asp AlaSer Thr Asn Arg Lys Arg Val Glu 500 505 510 Asn Thr Ala Lys Arg Lys LeuAsp Ser Leu Ile Lys Glu Ser Lys 515 520 525 Ile Arg Asp Cys Glu Asp ProAsn Asn Phe Ser Val Ser Thr Leu 530 535 540 Ser Pro Ser Gly Lys Leu GlyArg Lys Ser Lys Ala Glu Glu Asp 545 550 555 Val Glu Ser Gly Glu Asp AlaGly Ala Ser Arg Arg Asn Gly Arg 560 565 570 Leu Val Val Gly Ser Phe SerArg Arg Lys Lys Lys Gly Ser Lys 575 580 585 Leu Lys Lys Ala Ala Ser ValGlu Glu Gly Asp Glu Gly Gln Asp 590 595 600 Ser Pro Gly Gly Gln Ser ArgGly Ala Thr Arg Gln Lys Lys Thr 605 610 615 Met Lys Leu Ser Arg Ala LeuSer Asp Leu Val Lys Tyr Thr Lys 620 625 630 Ser Val Ala Thr His Asp IleGlu Met Glu Ala Ala Ser Ser Trp 635 640 645 Gln Val Ser Ser Phe Ser GluThr Lys Ala His Gln Ile Leu Gln 650 655 660 Gln Lys Pro Ala Gln Tyr LeuArg Phe Asn Gln Gln Gln Leu Ser 665 670 675 Arg Ile Tyr Pro Ser Ser TyrArg Val Asp Ser Ser Asn Tyr Asn 680 685 690 Pro Gln Pro Phe Trp Asn AlaGly Cys Gln Met Val Ala Leu Asn 695 700 705 Tyr Gln Ser Glu Gly Arg MetLeu Gln Leu Asn Arg Ala Lys Phe 710 715 720 Ser Ala Asn Gly Gly Cys GlyTyr Val Leu Lys Pro Gly Cys Met 725 730 735 Cys Gln Gly Val Phe Asn ProAsn Ser Glu Asp Pro Leu Pro Gly 740 745 750 Gln Leu Lys Lys Gln Leu ValLeu Arg Ile Ile Ser Gly Gln Gln 755 760 765 Leu Pro Lys Pro Arg Asp SerMet Leu Gly Asp Arg Gly Glu Ile 770 775 780 Ile Asp Pro Phe Val Glu ValGlu Ile Ile Gly Leu Pro Val Asp 785 790 795 Cys Ser Arg Glu Gln Thr ArgVal Val Asp Asp Asn Gly Phe Asn 800 805 810 Pro Thr Trp Glu Glu Thr LeuVal Phe Met Val His Met Pro Glu 815 820 825 Ile Ala Leu Val Arg Phe LeuVal Trp Asp His Asp Pro Ile Gly 830 835 840 Arg Asp Phe Ile Gly Gln ArgThr Leu Ala Phe Ser Ser Met Met 845 850 855 Pro Gly Tyr Arg His Val TyrLeu Glu Gly Met Glu Glu Ala Ser 860 865 870 Ile Phe Val His Val Ala ValSer Asp Ile Ser Gly Lys Val Lys 875 880 885 Gln Ala Leu Gly Leu Lys GlyLeu Phe Leu Arg Gly Pro Lys Pro 890 895 900 Gly Ser Leu Asp Ser His AlaAla Gly Arg Pro Pro Ala Arg Pro 905 910 915 Ser Val Ser Gln Arg Ile LeuArg Arg Thr Ala Ser Ala Pro Thr 920 925 930 Lys Ser Gln Lys Pro Gly ArgArg Gly Phe Pro Glu Leu Val Leu 935 940 945 Gly Thr Arg Asp Thr Gly SerLys Gly Val Ala Asp Asp Val Val 950 955 960 Pro Pro Gly Pro Gly Pro AlaPro Glu Ala Pro Ala Gln Glu Gly 965 970 975 Pro Gly Ser Gly Ser Pro ArgGly Lys Ala Pro Ala Ala Val Ala 980 985 990 Glu Lys Ser Pro Val Arg ValArg Pro Pro Arg Val Leu Asp Gly 995 1000 1005 Pro Gly Pro Ala Gly MetAla Ala Thr Cys Met Lys Cys Val Val 1010 1015 1020 Gly Ser Cys Ala GlyVal Asn Thr Gly Gly Pro Gln Arg Glu Arg 1025 1030 1035 Pro Pro Ser ProGly Pro Ala Ser Arg Gln Ala Ala Ile Arg Gln 1040 1045 1050 Gln Pro ArgAla Arg Ala Asp Ser Leu Gly Ala Pro Cys Cys Gly 1055 1060 1065 Leu AspPro His Ala Ile Pro Gly Arg Ser Arg Glu Ala Pro Lys 1070 1075 1080 GlyPro Gly Ala Trp Arg Gln Gly Pro Gly Gly Ser Gly Ser Met 1085 1090 1095Ser Ser Asp Ser Ser Ser Pro Asp Ser Pro Gly Ile Pro Glu Arg 1100 11051110 Ser Pro Arg Trp Pro Glu Gly Ala Cys Arg Gln Pro Gly Ala Leu 11151120 1125 Gln Gly Glu Met Ser Ala Leu Phe Ala Gln Lys Leu Glu Glu Ile1130 1135 1140 Arg Ser Lys Ser Pro Met Phe Ser Ala Val Arg Asn 1145 11507 1294 PRT Homo sapiens misc_feature Incyte ID No 6131380CD1 7 Met IleIle Lys Glu Tyr Arg Ile Pro Leu Pro Met Thr Val Glu 1 5 10 15 Glu TyrArg Ile Ala Gln Leu Tyr Met Ile Gln Lys Lys Ser Arg 20 25 30 Asn Glu ThrTyr Gly Glu Gly Ser Gly Val Glu Ile Leu Glu Asn 35 40 45 Arg Pro Tyr ThrAsp Gly Pro Gly Gly Ser Gly Gln Tyr Thr His 50 55 60 Lys Val Tyr His ValGly Met His Ile Pro Ser Trp Phe Arg Ser 65 70 75 Ile Leu Pro Lys Ala AlaLeu Arg Val Val Glu Glu Ser Trp Asn 80 85 90 Ala Tyr Pro Tyr Thr Arg ThrArg Phe Thr Cys Pro Phe Val Glu 95 100 105 Lys Phe Ser Ile Asp Ile GluThr Phe Tyr Lys Thr Asp Ala Gly 110 115 120 Glu Asn Pro Asp Val Phe AsnLeu Ser Pro Val Glu Lys Asn Gln 125 130 135 Leu Thr Ile Asp Phe Ile AspIle Val Lys Asp Pro Val Pro His 140 145 150 Asn Glu Tyr Lys Thr Glu GluAsp Pro Lys Leu Phe Gln Ser Thr 155 160 165 Lys Thr Gln Arg Gly Pro LeuSer Glu Asn Trp Ile Glu Glu Tyr 170 175 180 Lys Lys Gln Val Phe Pro IleMet Cys Ala Tyr Lys Leu Cys Lys 185 190 195 Val Glu Phe Arg Tyr Trp GlyMet Gln Ser Lys Ile Glu Arg Phe 200 205 210 Ile His Asp Thr Gly Leu ArgArg Val Met Val Arg Ala His Arg 215 220 225 Gln Ala Trp Cys Trp Gln AspGlu Trp Tyr Gly Leu Ser Met Glu 230 235 240 Asn Ile Arg Glu Leu Glu LysGlu Ala Gln Leu Met Leu Ser Arg 245 250 255 Lys Met Ala Gln Phe Asn GluAsp Gly Glu Glu Ala Thr Glu Leu 260 265 270 Val Lys His Glu Ala Val SerAsp Gln Thr Ser Gly Glu Pro Pro 275 280 285 Glu Pro Ser Ser Ser Asn GlyGlu Pro Leu Val Gly Arg Gly Leu 290 295 300 Lys Lys Gln Trp Ser Thr SerSer Lys Ser Ser Arg Ser Ser Lys 305 310 315 Arg Gly Ala Ser Pro Ser ArgHis Ser Ile Ser Glu Trp Arg Met 320 325 330 Gln Ser Ile Ala Arg Asp SerAsp Glu Ser Ser Asp Asp Glu Phe 335 340 345 Phe Asp Ala His Glu Asp LeuSer Asp Thr Glu Glu Met Phe Pro 350 355 360 Lys Asp Ile Thr Lys Trp SerSer Asn Asp Leu Met Asp Lys Ile 365 370 375 Glu Ser Pro Glu Pro Glu AspThr Gln Asp Gly Leu Tyr Arg Gln 380 385 390 Gly Ala Pro Glu Phe Arg ValAla Ser Ser Val Glu Gln Leu Asn 395 400 405 Ile Ile Glu Asp Glu Val SerGln Pro Leu Ala Ala Pro Pro Ser 410 415 420 Lys Ile His Val Leu Leu LeuVal Leu His Gly Gly Thr Ile Leu 425 430 435 Asp Thr Gly Ala Gly Asp ProSer Ser Lys Lys Gly Asp Ala Asn 440 445 450 Thr Ile Ala Asn Val Phe AspThr Val Met Arg Val His Tyr Pro 455 460 465 Ser Ala Leu Gly Arg Leu AlaIle Arg Leu Val Pro Cys Pro Pro 470 475 480 Val Cys Ser Asp Ala Phe AlaLeu Val Ser Asn Leu Ser Pro Tyr 485 490 495 Ser His Asp Glu Gly Cys LeuSer Ser Ser Gln Asp His Ile Pro 500 505 510 Leu Ala Ala Leu Pro Leu LeuAla Thr Ser Ser Pro Gln Tyr Gln 515 520 525 Glu Ala Val Ala Thr Val IleGln Arg Ala Asn Leu Ala Tyr Gly 530 535 540 Asp Phe Ile Lys Ser Gln GluGly Met Thr Phe Asn Gly Gln Val 545 550 555 Cys Leu Ile Gly Asp Cys ValGly Gly Ile Leu Ala Phe Asp Ala 560 565 570 Leu Cys Tyr Ser Asn Gln ProVal Ser Glu Ser Gln Ser Ser Ser 575 580 585 Arg Arg Gly Ser Val Val SerMet Gln Asp Asn Asp Leu Leu Ser 590 595 600 Pro Gly Ile Leu Met Asn AlaAla His Cys Cys Gly Gly Gly Gly 605 610 615 Gly Gly Gly Gly Gly Gly GlySer Ser Gly Gly Gly Gly Ser Ser 620 625 630 Gly Gly Ser Ser Leu Glu SerSer Arg His Leu Ser Arg Ser Asn 635 640 645 Val Asp Ile Pro Arg Ser AsnGly Thr Glu Asp Pro Lys Arg Gln 650 655 660 Leu Pro Arg Lys Arg Ser AspSer Ser Thr Tyr Glu Leu Asp Thr 665 670 675 Ile Gln Gln His Gln Ala PheLeu Ser Ser Leu His Ala Ser Val 680 685 690 Leu Arg Thr Glu Pro Cys SerArg His Ser Ser Ser Ser Thr Met 695 700 705 Leu Asp Gly Thr Gly Ala LeuGly Arg Phe Asp Phe Glu Ile Thr 710 715 720 Asp Leu Phe Leu Phe Gly CysPro Leu Gly Leu Val Leu Ala Leu 725 730 735 Arg Lys Thr Val Ile Pro AlaLeu Asp Val Phe Gln Leu Arg Pro 740 745 750 Ala Cys Gln Gln Val Tyr AsnLeu Phe His Pro Ala Asp Pro Ser 755 760 765 Ala Ser Arg Leu Glu Pro LeuLeu Glu Arg Arg Phe His Ala Leu 770 775 780 Pro Pro Phe Ser Val Pro ArgTyr Gln Arg Tyr Pro Leu Gly Asp 785 790 795 Gly Cys Ser Thr Leu Leu AspVal Leu Gln Thr His Asn Ala Ala 800 805 810 Phe Gln Glu His Gly Ala ProSer Ser Pro Gly Thr Ala Pro Ala 815 820 825 Ser Arg Gly Phe Arg Arg AlaSer Glu Ile Ser Ile Ala Ser Gln 830 835 840 Val Ser Gly Met Ala Glu SerTyr Thr Ala Ser Ser Ile Ala Gln 845 850 855 Val Ala Ala Lys Trp Trp GlyGln Lys Arg Ile Asp Tyr Ala Leu 860 865 870 Tyr Cys Pro Asp Ala Leu ThrAla Phe Pro Thr Val Ala Leu Pro 875 880 885 His Leu Phe His Ala Ser TyrTrp Glu Ser Thr Asp Val Val Ser 890 895 900 Phe Leu Leu Arg Gln Val MetArg His Asp Asn Ser Ser Ile Leu 905 910 915 Glu Leu Asp Gly Lys Glu ValSer Val Phe Thr Pro Ser Lys Pro 920 925 930 Arg Glu Lys Trp Gln Arg LysArg Thr His Val Lys Leu Arg Asn 935 940 945 Val Thr Ala Asn His Arg IleAsn Asp Ala Leu Ala Asn Glu Asp 950 955 960 Gly Pro Gln Val Leu Thr GlyArg Phe Met Tyr Gly Pro Leu Asp 965 970 975 Met Val Thr Leu Thr Gly GluLys Val Asp Val His Ile Met Thr 980 985 990 Gln Pro Pro Ser Gly Glu TrpLeu Tyr Leu Asp Thr Leu Val Thr 995 1000 1005 Asn Asn Ser Gly Arg ValSer Tyr Thr Ile Pro Glu Ser His Arg 1010 1015 1020 Leu Gly Val Gly ValTyr Pro Ile Lys Met Val Val Arg Gly Asp 1025 1030 1035 His Thr Phe AlaAsp Ser Tyr Ile Thr Val Leu Pro Lys Gly Thr 1040 1045 1050 Glu Phe ValVal Phe Ser Ile Asp Gly Ser Phe Ala Ala Ser Val 1055 1060 1065 Ser IleMet Gly Ser Asp Pro Lys Val Arg Ala Gly Ala Val Asp 1070 1075 1080 ValVal Arg His Trp Gln Asp Leu Gly Tyr Leu Ile Ile Tyr Val 1085 1090 1095Thr Gly Arg Pro Asp Met Gln Lys Gln Arg Val Val Ala Trp Leu 1100 11051110 Ala Gln His Asn Phe Pro His Gly Val Val Ser Phe Cys Asp Gly 11151120 1125 Leu Val His Asp Pro Leu Arg His Lys Ala Asn Phe Leu Lys Leu1130 1135 1140 Leu Ile Ser Glu Leu His Leu Arg Val His Ala Ala Tyr GlySer 1145 1150 1155 Thr Lys Asp Val Ala Val Tyr Ser Ala Ile Ser Leu SerPro Met 1160 1165 1170 Gln Ile Tyr Ile Val Gly Arg Pro Thr Lys Lys LeuGln Gln Gln 1175 1180 1185 Cys Gln Phe Ile Thr Asp Gly Tyr Ala Ala HisLeu Ala Gln Leu 1190 1195 1200 Lys Tyr Ser His Arg Ala Arg Pro Ala ArgAsn Thr Ala Thr Arg 1205 1210 1215 Met Ala Leu Arg Lys Gly Ser Phe GlyLeu Pro Gly Gln Gly Asp 1220 1225 1230 Phe Leu Arg Ser Arg Asn His LeuLeu Arg Thr Ile Ser Ala Gln 1235 1240 1245 Pro Ser Gly Pro Ser His ArgHis Glu Arg Thr Gln Ser Gln Ala 1250 1255 1260 Asp Gly Glu Gln Arg GlyGln Arg Ser Met Ser Val Ala Ala Gly 1265 1270 1275 Cys Trp Gly Arg AlaMet Thr Gly Arg Leu Glu Pro Gly Ala Ala 1280 1285 1290 Ala Gly Pro Lys 877 PRT Homo sapiens misc_feature Incyte ID No 643681CD1 8 Met Asp MetVal Arg Trp Cys Gly Glu Asp Val Arg Lys Leu Glu 1 5 10 15 Val Phe IleThr Ser Gln Gly Ala Ser Glu Tyr Arg Gly Lys Lys 20 25 30 Thr Thr Lys ArgGln Ala Gln Gly Glu Ser Thr Ile Lys Asp Ile 35 40 45 Pro Met Pro Ala SerIle Ala Ala Pro Ala Leu Leu Ala Gly His 50 55 60 Leu Pro Gln Leu His LeuPro Ser Lys Leu Phe Asn Phe His Thr 65 70 75 Val Ser 9 576 PRT Homosapiens misc_feature Incyte ID No 6897474CD1 9 Met Ala Gln Gly Val LeuTrp Ile Leu Leu Gly Leu Leu Leu Trp 1 5 10 15 Ser Asp Pro Gly Thr AlaSer Leu Pro Leu Leu Met Asp Ser Val 20 25 30 Ile Gln Ala Leu Ala Glu LeuGlu Gln Lys Val Pro Ala Ala Lys 35 40 45 Thr Arg His Thr Ala Ser Ala TrpLeu Met Ser Ala Pro Asn Ser 50 55 60 Gly Pro His Asn Arg Leu Tyr His PheLeu Leu Gly Ala Trp Ser 65 70 75 Leu Asn Ala Thr Glu Leu Asp Pro Cys ProLeu Ser Pro Glu Leu 80 85 90 Leu Gly Leu Thr Lys Glu Val Ala Arg His AspVal Arg Glu Gly 95 100 105 Lys Glu Tyr Gly Val Val Leu Ala Pro Asp GlySer Thr Val Ala 110 115 120 Val Glu Pro Leu Leu Ala Gly Leu Glu Ala GlyLeu Gln Gly Arg 125 130 135 Arg Val Ile Asn Leu Pro Leu Asp Ser Met AlaAla Pro Trp Glu 140 145 150 Thr Gly Asp Thr Phe Pro Asp Val Val Ala IleAla Pro Asp Val 155 160 165 Arg Ala Thr Ser Ser Pro Gly Leu Arg Asp GlySer Pro Asp Val 170 175 180 Thr Thr Ala Asp Ile Gly Ala Asn Thr Pro AspAla Thr Lys Gly 185 190 195 Cys Pro Asp Val Gln Ala Ser Leu Pro Asp AlaLys Ala Lys Ser 200 205 210 Pro Pro Thr Met Val Asp Ser Leu Leu Ala ValThr Leu Ala Gly 215 220 225 Asn Leu Gly Leu Thr Phe Leu Arg Gly Ser GlnThr Gln Ser His 230 235 240 Pro Asp Leu Gly Thr Glu Gly Cys Trp Asp GlnLeu Ser Ala Pro 245 250 255 Arg Thr Phe Thr Leu Leu Asp Pro Lys Ala SerLeu Leu Thr Met 260 265 270 Ala Phe Leu Asn Gly Ala Leu Asp Gly Val IleLeu Gly Asp Tyr 275 280 285 Leu Ser Arg Thr Pro Glu Pro Arg Pro Ser LeuSer His Leu Leu 290 295 300 Ser Gln Tyr Tyr Gly Ala Gly Val Ala Arg AspPro Gly Phe Arg 305 310 315 Ser Asn Phe Arg Arg Gln Asn Gly Ala Ala LeuThr Ser Ala Ser 320 325 330 Ile Leu Ala Gln Gln Val Trp Gly Thr Leu ValLeu Leu Gln Arg 335 340 345 Leu Glu Pro Val His Leu Gln Leu Gln Cys MetSer Gln Glu Gln 350 355 360 Leu Ala Gln Val Ala Ala Asn Ala Thr Lys GluPhe Thr Glu Ala 365 370 375 Phe Leu Gly Cys Pro Ala Ile His Pro Arg CysArg Trp Gly Ala 380 385 390 Ala Pro Tyr Arg Gly Arg Pro Lys Leu Leu GlnLeu Pro Leu Gly 395 400 405 Phe Leu Tyr Val His His Thr Tyr Val Pro AlaPro Pro Cys Thr 410 415 420 Asp Phe Thr Arg Cys Ala Ala Asn Met Arg SerMet Gln Arg Tyr 425 430 435 His Gln Asp Thr Gln Gly Trp Gly Asp Ile GlyTyr Ser Phe Val 440 445 450 Val Gly Ser Asp Gly Tyr Val Tyr Glu Gly ArgGly Trp His Trp 455 460 465 Val Gly Ala His Thr Leu Gly His Asn Ser ArgGly Phe Gly Val 470 475 480 Ala Ile Val Gly Asn Tyr Thr Ala Ala Leu ProThr Glu Ala Ala 485 490 495 Leu Arg Thr Val Arg Asp Thr Leu Pro Ser CysAla Val Arg Ala 500 505 510 Gly Leu Leu Arg Pro Asp Tyr Ala Leu Leu GlyHis Arg Gln Leu 515 520 525 Val Arg Thr Asp Cys Pro Gly Asp Ala Leu PheAsp Leu Leu Arg 530 535 540 Thr Trp Pro His Phe Thr Ala Thr Val Lys ProArg Pro Ala Arg 545 550 555 Ser Val Ser Lys Arg Ser Arg Arg Glu Pro ProPro Arg Thr Leu 560 565 570 Pro Ala Thr Asp Leu Gln 575 10 3879 DNA Homosapiens misc_feature Incyte ID No 7472774CB1 10 aggtccctgg ccacagctcctggggtacca agccatgaaa ctgaagtgga gttgggagcg 60 acggtcgcat cctcctagaggggcatctat gagccatgac ctctataagc tgaagagata 120 gagctttccc aaattatggcgggctagtcc tacagtcatg tgggtccagt gtcctcttct 180 tgccacccac tgtgcccttgaaggcctggt cattctgagt ggctgggggc tacagactgc 240 tgaccccaaa gaccagagccctgcgggtcc ctgtatttct atgacctgaa gacctgtgat 300 ttctttgata tgaagagatctaggcccatg caccctatct gtctacccac tcaaaccact 360 cccagagcaa tcccagctactgccaagctg tggccaggaa ggtggagctc tgagtcagag 420 tataagttcc tgatcttgccacccagctgg agagctgccg tgatgctcct gaggcagatg 480 cacgccaggg tctcccactccctgccagac ccatgccaag cagaagacag caggccctcg 540 gccacctgtg ccttgaaggctccccagact tcatgggatg gtttgctgag ggaggggctg 600 tctccatgcc acctgttgacagtgagggtc atccggatga aaaatgtccg gcaggctgat 660 atgcaaccag taggtatagagctggcaccc tgcctgcagg ctcccagcgt accggagaca 720 gacctgaagg gtgtggtccaggcccggggt gggggggcca gtgttctgga aaagccaagg 780 gaagggttca agagggctgagcaggttcct gtgagccaga cagactgttt tgtgagcctc 840 tggctgccca ccgcctctcagaagaagctg aggacaagga ccatctccaa ctgcccaaat 900 ccagagtgga atgaaagcttcaacttccag atccagagcc gagtgaagaa cgtgctagag 960 ttgagtgtct gtgatgaagacacagtgaca ccagatgacc atctcctgac agttctctat 1020 gacctcacca agctctgtttccgaaagaaa acccacgtga agtttccact caacccgcag 1080 ggcatggaag agctggaggtggagttcctg ctggaggaga gtccctctcc acctgagacc 1140 ctcgtcacca atggcgtgctggtggtaatt atcttcctgg gttcctgtag ctccagaggc 1200 cacggctggc tgctgctctcaggggaacag gaccaaggga gaaaacagtg ggcccagctt 1260 ggtctctgtc ctatcctgacctctgcagga gttagactaa acgaggccag ccaaatgggg 1320 cacaggcagc actggggcacgagctggggc ttctgtacag agggaggagt gaaggacctc 1380 ctggtgatgg tgaacgaatcctttgagaac acccagcgtg tccggccctg cttggaaccc 1440 tgctgcccaa cctctgcctgcttccaaacc gctgcctgct tccactaccc caagtacttc 1500 cagtcccagg tgcacgtggaagtgcccaag agtcactgga gctgtgggct ttgctgccgc 1560 tctcgcaaga agggccccatcagccagccc ctcgactgcc tttccgatgg tcaggtgatg 1620 accctgcctg tgggtgagagttatgaatta cacatgaagt ctacaccctg ccctgagaca 1680 ctggacgtgc ggctgggcttcagcctgtgc ccagcagagc tggagtttct gcagaagcgg 1740 aaggtcgtgg tggccaaggccctgaagcag gtgctgcagc tggaggaaga cctgcaggag 1800 gacgaggtgc cgctgatagccatcatggcc actgggggtg gaacaagatc catgacctcc 1860 atgtatggcc acctgctggggctgcagaag ctgaacctcc tggactgtgc cagctacatc 1920 actggtctat caggggccacctggaccatg gctaccttgt accgtgaccc tgactggtcc 1980 tccaaaaact tggagcctgctatctttgag gctcggagac atgtggtaaa ggacaagcta 2040 ccctccctgt tcccagaccagctccgcaaa ttccaggagg agctccggca gcgcagccag 2100 gaaggctaca gggtcacctttacagacttc tggggcctgc tgatagagac ctgcctgggg 2160 gacgagagaa atgaatgcaaactgtcagat cagcgtgctg ctttgagctg cggccagaac 2220 cccctgccca tctacctcaccatcaatgtc aaggatgatg taagcaacca ggatgtcaga 2280 tggttcgagt tctccccctacgaggtgggc ctgcagaagt atggggcctt catcccctcc 2340 gagctcttcg gctccgagttcttcatgggg cggctggtga agaggatccc ggagtctcga 2400 atctgctaca tgctaggcctgtggagcagc atcttctccc tgaacctgct ggatgcctgg 2460 aacctgtcac acacctcggaggagtttttc cacaggtgga caagggagaa agtgcaggac 2520 atcgaagacg agccgatcctgcctgaaatc cccaaatgtg atgctaacat cctggagacc 2580 acggtagtga tcccagggtcatggctgtcc aattctttcc gagaaatcct tacccatcgg 2640 tccttcgtgt ctgagtttcacaacttcctg tctgggctgc agctgcacac caactacctc 2700 cagaatggcc agttctctaggtggaaagac acagtgctag atggtttccc aaaccagctg 2760 accgagtccg cgaaccacctgtgcctgctg gacactgcgt tctttgtcaa ctccagctac 2820 ccgcccctcc tcaggccagagcgaaaagcc gatctcatca tccacctcaa ctactgtgct 2880 gggtcccaga caaagcccctgaaacaaacc tgtgagtact gcactgtgca gaacatcccc 2940 ttccccaaat acgagctgccagatgagaat gaaaatctca aggaatgcta cctgatggag 3000 aacccccagg aacccgatgcccccatcgtg actttcttcc cactcatcaa tgacactttc 3060 cgaaaataca aggcaccaggtgtagagcga agccctgagg agctggagca gggccaggtg 3120 gacatttatg gtcccaaaactccctatgcc accaaggagc tgacatacac agaggccacc 3180 tttgacaagc tggtgaaactctcagagtat aacatcctga ataataagga cactctcctc 3240 caggctctgc ggctcgcagtggagaagaag aagcgcctga agggccagtg tccctcctag 3300 gccccaggga gcctcccctgttctgtgtca gcttctacca tcagaggtgc aggacccctc 3360 agggctgacc aggttactacgcagccagct ctgctctccg gcaatgggtg tgagcaggtt 3420 ggcctgggct ttctaacgaaaagtaaaaaa ttttaaaaag ttgagaaagt cagaaagaga 3480 gagagaggag ctctgttggggttttatacc cactagagtt tcttcaagtg cttccctata 3540 gagaaggtgg tctcatagccacaggctccc acacatctgt ggagaggaaa agcctgggga 3600 agaggctggg cccccagaaacctcgactca gaggcagagc ccagggctgg cagccctcct 3660 ctctctgtcc tctacctcgtgtggcgggcc tagggaaatg cacagaagga cctgagaggc 3720 actcggcgtt tcactggaaaaacacttcaa aatttaaggc aattctagtc ttgtgatttt 3780 tggttttttt tagacggagtctcactctgt tgcccaggct ggagtgcaat ggcgcgatct 3840 cggctcactg caacctctgcctcccaggtt caagcaatt 3879 11 1623 DNA Homo sapiens misc_feature IncyteID No 2884821CB1 11 gcttggctgc ttgtcataaa tggagcgacg taatttcgacctgtcctttc ccgggagtta 60 gcgatccctc aacccctgca ctgcgctagt cctaaagaggaaatgtctct acgctgcggg 120 gatgcagccc gcaccctggg gccccgggta tttgggagatatttttgcag cccagtcaga 180 ccgttaagct ccttgccaga taaaaaaaag gaactcctacagaatggacc agaccttcaa 240 gattttgtat ctggtgatct tgcagacagg agcacctgggatgaatataa aggaaaccta 300 aaacgccaga aaggagaaag gttaagacta cctccatggctaaagacaga gattcccatg 360 gggaaaaatt acaataaact gaaaaatact ttgcggaatttaaatctcca tacagtatgt 420 gaggaagctc gatgtcccaa tattggagag tgttggggaggtggagaata tgccaccgcc 480 acagccacga tcatgttgat gggtgacaca tgtacaagaggttgcagatt ttgttctgtt 540 aagactgcaa gaaatcctcc tccactggat gccagtgagccctacaatac tgcaaaggca 600 attgcagaat ggggtctgga ttatgttgtc ctgacatctgtggatcgaga tgatatgcct 660 gatgggggag ctgaacacat tgcaaagacc gtatcatatttaaaggaaag gaatccaaaa 720 atccttgtgg agtgtcttac tcctgatttt cgaggtgatctcaaagcaat agaaaaagtt 780 gctctgtcag gattagatgt gtatgcacat aatgtagaaacagtcccgga attacagagt 840 aaggttcgtg atcctcgggc caattttgat cagtccctacgtgtactgaa acatgccaag 900 aaggttcagc ctgatgttat ttctaaaaca tctataatgttgggtttagg cgagaatgat 960 gagcaagtat atgcaacaat gaaagcactt cgtgaggcagatgtagactg cttgacttta 1020 ggacaatata tgcagccaac aaggcgtcac cttaaggttgaagaatatat tactcctgaa 1080 aaattcaaat actgggaaaa agtaggaaat gaacttggatttcattatac tgcaagtggc 1140 cctttggtgc gttcttcata taaagcaggt gaatttttcctgaaaaatct agtggctaaa 1200 agaaaaacaa aagacctcta aaacttcaac aagaccttcaagatcacaga aatttttaaa 1260 atttgattcc agttaataac agaggtggtg ccagaatgcctggactgcag tggatgtacc 1320 ccacctcttt gcttaaaaaa aaaaatgtca atagccaggcatagtggctc acgcctgtaa 1380 tcccagcact ttaggaggcc aaggcgggtg gatcacctgaggtcaggagt tcgagaccag 1440 cctggccaac atggtgaaat cctgtctcca ctaaaaacacaaaaattagt caggcgtggt 1500 agtgggtgcc tgtaatccca gctactcggg aggctaaggcaggagaatca cttgaacctg 1560 ggagggggag gttgcagtga gccaagatcg ctccattgccctccagcctg ggtgacaaga 1620 gca 1623 12 2199 DNA Homo sapiensmisc_feature Incyte ID No 72852842CB1 12 cagctatacc tcttttgaagattttaagaa cttagcctcc tgaacagtct tcttcgaaag 60 tgaaaagtgg taacagctgatgagtatcaa gaaattattt tctgcaaagg ggcagagtta 120 attgtatttg gaacccatgacagcacctac tggggaaaga cttctaagtg aggagaaacg 180 gctctacagg tcatgaaactatggaaatga gatggttttt gtcaaagatt caggatgact 240 tcagaggtgg aaaaattaacctagaaaaaa ctcagaggtt acttgaaaaa ttagatattc 300 ggtgcagtta tattcatgtgaaacagattt ttaaggacaa tgacaggctg aaacaaggaa 360 gaatcaccat agaagaatttagagcaattt atcgaattat cacgcacaga gaagaaatta 420 ttgagatttt caacacatattctgaaaacc ggaaaattct tttagcaagt aatctggctc 480 aatttctgac acaagaacaatatgcagctg agatgagtaa agctattgct tttgagatca 540 ttcagaaata cgagcctatcgaagaagtta ggaaagcaca ccaaatgtca ttagaaggtt 600 ttacaagata catggattcacgtgaatgtc tactgtttaa aaatgaatgt agaaaagttt 660 atcaagatat gactcatccattaaatgatt attttatttc atcttcacat aacacatatt 720 tggtatctga tcaattattgggaccaagtg acctttgggg atatgtaagt gcccttgtga 780 aaggatgccg ttgtttggagattgactgct gggatggagc acaaaatgaa cctgttgtat 840 atcatggcta cacactcacaagcaaacttc tgtttaaaac tgttatccaa gctatacaca 900 agtatgcatt catgacatctgactacccag tggtgctctc tttagaaaat cactgctcca 960 ctgcccaaca agaagtaatggcagacaatt tgcaggctac ttttggagag tccttgcttt 1020 ctgatatgct tgatgattttcctgatactc taccatcacc agaggcacta aaattcaaaa 1080 tattagttaa aaataagaaaataggaacct taaaggaaac ccatgaaaga aaaggttctg 1140 ataagcgtgg taaggtggaggaatgggaag aagaagtggc agatggagag gaggaggagg 1200 aggaggagga ggaggaggaggaggaggagg aggataaatt caaagaatca gaagtattgg 1260 aatctgtttt aggagacaatcaagacaagg aaacaggggt aaaaaagtta cctggagtaa 1320 tgcttttcaa gaaaaagaagaccaggaagc taaaaattgc tctggcctta tctgatcttg 1380 tcatttatac gaaagctgagaaattcaaaa gctttcaaca ttcaagatta tatcagcaat 1440 ttaatgaaaa taattctattggggagacac aagcccgaaa actttcaaaa ttgcgagtcc 1500 atgagtttat ttttcacaccaggaagttca ttaccagaat atatcccaaa gcaacaagag 1560 cagactcttc taattttaatccccaagaat tttggaatat aggttgtcaa atggtggctt 1620 taaatttcca gacccctggtctgcccatgg atctgcaaaa tgggaaattt ttggataatg 1680 gtggttctgg atatattttgaaaccacatt tcttaagaga gagtaaatca tactttaacc 1740 caagtaacat aaaagagggtatgccaatta cacttacaat aaggctcatc agtggtatcc 1800 agttgcctct tactcattcatcatctaaca aaggtgattc attagtaatt atagaagttt 1860 ttggtgttcc aaatgatcaaatgaagcagc agactcgtgt aattaaaaaa aatgctttta 1920 gtccaagatg gaatgaaacattcacattta ttattcatgt cccagaattg gcattgatac 1980 gttttgttgt tgaaggtcaaggtttaatag caggaaatga atttcttggg caatatactt 2040 tgccacttct atgcatgaacaaaggttatc gtcgtattcc tctgttttcc agaatgggtg 2100 agagccttga gcctgcttcactgtttgttt atgtttggta cgtcagataa cagctaatga 2160 taaatgacat atcattagctatgcatcgca ataaaaccg 2199 13 6326 DNA Homo sapiens misc_feature IncyteID No 7484271CB1 13 aggaagggga ggagggggtg ggatattatg atgcagctcacattgaacac tctctttcct 60 gttgtttcca caccagctat tacgtatatt gtcaccgtcttcactgggga tgtccggggg 120 gctggtacca atatacgtaa tagctggtgt tgtaaccaggaaggggagga gggggtggga 180 tattatgatg cagctcacat tgaacactct ctttcctgttgtttccacac cagctattac 240 gtatattgtc accgtcttca ctggggatgt ccggggggctggtaccaaat ccaaaatcta 300 cttggtcatg tatggggcca gagggaataa gaacagtgggaaaatcttcc tggagggcgg 360 cgtgtttgac cgaggccgca cggacatctt ccacatcgagctggctgtcc tccttagccc 420 cctgagtcgg gtctccgtcg ggcatggcaa tgtgggtgtcaacagaggct ggttctgtga 480 gaaggtggtg attctgtgcc ccttcactgg tatccagcagaccttccctt gtagcaactg 540 gctggatgag aagaaagcgg atgggttgat cgagaggcagctctatgaga tggtgtctct 600 caggaagaag cggctgaaaa aattcccttg gtccctgtgggtctggacaa ccgacctaaa 660 gaaagctggt accaactctc ccatcttcat ccagatttatgggcagaagg ggcggacaga 720 tgagattctc ctgaatccca acaacaagtg gttcaaacccggcataatcg agaagtttag 780 gattgagctc ccggatcttg gcaggtttta taagattcgagtatggcatg ataaaaggag 840 ttctggttct ggatggcatt tagaaaggat gaccctgatgaacactctga acaaagacaa 900 gtacaacttc aattgcaacc gctggctgga tgccaatgaggatgacaatg agatagtgag 960 ggaaatgact gcagaaggcc caacagtgcg caggatcatgggcatggccc ggtaccatgt 1020 gactgtgtgc acaggtgaac ttgaaggtgc tgggaccgatgccaacgtct atctctgcct 1080 ttttggtgat gtgggggaca cgggggaacg gctgctctacaactgcagga ataacacaga 1140 cctgtttgaa aagggcaatg ctgacgagtt cactatcgagtctgtcacca tgcggaatgt 1200 gaggcgggtg aggatcagac acgatggcaa aggctccggcagcggctggt acctggacag 1260 agtgctggtg agagaggagg ggcagcctga gagcgacaacgtggagttcc catgtctcag 1320 gtggttggac aaggataagg atgatgggca gctggtccgagagttgctac ccagtgacag 1380 cagcgcgaca ctgaagaact ttcgctatca catcagcttgaagactgggg atgtctctgg 1440 ggccagcacg gattctagag tctacatcaa gctctatggggataaatctg acaccatcaa 1500 gcaagttctt cttgtctctg acaacaacct caaagactactttgaacgtg gccgggtgga 1560 tgagttcacc ctcgagaccc tgaacattgg aaatatcaaccggctggtga ttgggcatga 1620 cagcactggc atgcatgcca gctggttcct gggcagcgttcagatccgtg tgccccgtca 1680 aggcaagcag tacacctttc ccgccaaccg ctggctggacaagaaccagg ctgacgggcg 1740 cctggaggtg gagctgtatc ccagcgaggt ggtggagatccagaaattgg tccactatga 1800 ggttgagatt tggacaggag atgtgggtgg cgcaggcaccagtgcccgag tctacatgca 1860 gatctatgga gagaaaggca agacagaagt gctcttcctctccagccgct caaaagtttt 1920 tgaacgggcg tccaaggaca cattccagct tgaggcggccgacgtgggcg aggtctataa 1980 gctccggctc gggcacacgg gcgagggctt tgggcccagctggttcgtgg acaccgtgtg 2040 gctgcggcac ctggtggtgc gggaggtgga cctcacgccggaggaggagg cccggaagaa 2100 gaaggagaag gacaagctgc ggcagctgct caagaaggagcggctgaagg ccaagctgca 2160 gaggaagaag aagaagagga agggcagcga cgaagaggacgagggggagg aagaggagtc 2220 gtcctcatca gaggagtcct cgtcagagga ggaggagatggaagaagagg aggaagagga 2280 ggagtttggg ccggggatgc aggaggtgat tgagcagcacaagttcgaag cccaccgctg 2340 gctggcccgg ggcaaggagg acaacgaact tgtcgtggagttggtgccag ctggcaagcc 2400 gggtcctgag cgaaacacct atgaggttca ggtggtcacggggaatgtgc ccaaggccgg 2460 cactgatgct aacgtctacc taaccatcta cggcgaggagtatggagaca cgggcgaacg 2520 acccctgaag aagtcagaca agtccaacaa atttgagcaggggcagacag acaccttcac 2580 catctatgcc attgacctgg gggccctgac caagattcggattcgccacg acaacacagg 2640 caacagagca ggctggttcc tggacagaat agacattactgacatgaaca acgagatcac 2700 gtactacttt ccatgccaac gttggctggc agtggaggaagatgatggcc agctgtccag 2760 ggagctgttg ccagtggatg agtcctatgt gctgccacagagcgaggagg gtgggggagg 2820 cggtgacaac aaccccctcg acaacctggc cctggagcagaaagataaat ctaccacatt 2880 ctcagtgacc ataaagactg gggttaagaa gaatgcgggcacagatgcta atgtcttcat 2940 cacactcttt ggcacacagg atgacactgg aatgaccctcctgaagtcct ccaagacaaa 3000 cagcgataag tttgagaggg acagcattga aatcttcacggtggagacgc tggatctggg 3060 agacctgtgg aaagtccggc ttggccatga caacacaggcaaggccccag gctggtttgt 3120 agactgggta gaggtggatg ccccatctct tgggaagtgcatgacgtttc cctgtggccg 3180 ctggctggcc aaaaacgaag acgacgggtc catcatcagagacctcttcc atgcagagct 3240 tcagacgagg ctgtacacac catttgttcc ttacgagatcaccctctaca ccagtgatgt 3300 ctttgctgct gggacagatg ccaacatctt catcatcatctatggctgcg atgccgtgtg 3360 cacccagcag aagtatctgt gtaccaacaa gagggaacagaagcagttct ttgagaggaa 3420 gtctgcctcc cgcttcatcg tagagttaga agatgtgggagaaatcattg aaaaaattcg 3480 gattggccat aataacacgg gcatgaatcc tgggtggcactgctctcacg tggacatccg 3540 caggctcctc ccggataaag acggtgcaga gaccttgactttcccatgcg atcggtggct 3600 tgccacctct gaggatgaca aaaagaccat tcgagaactggttccatatg acatcttcac 3660 tgagaaatac atgaaagatg ggtccttacg gcaagtctacaaggaagtag aagagcctct 3720 ggacattgtg ctgtactcgg tgcagatctt cacagggaacattcctgggg cagggacgga 3780 tgccaaggtg tacatcacca tctatggaga cctcggggacactggggagc gataccttgg 3840 caagtcagag aaccggacca acaagttcga gagaggaacggctgacacct tcatcatcga 3900 ggccgctgac ctaggcgtca tctacaagat caagctccgccatgacaact ccaagtggtg 3960 cgcagactgg tacgtggaga aggtggagat ctggaatgacaccaacgagg acgagttcct 4020 gttcctatgc gggcgctggc tctccctgaa gaaggaggatgggcgactcg agaggctctt 4080 ttacgagaag gagtacactg gggaccgcag cagcaactgcagcagccctg ctgacttctg 4140 ggagatcgcc ctgagctcca agatggccga tgtcgacatcagcacagtga ccgggcccat 4200 ggctgactac gttcaagagg gcccaattat tccctactatgtgtcagtca ccactgggaa 4260 gcacaaggac gcggccactg acagccgagc cttcatctttctcatcgggg aggatgatga 4320 acgtagtaag cgcatctggt tggactaccc ccgagggaagaggggcttca gccgtggctc 4380 tgtggaggag ttctacgtcg caggcttgga tgtgggcatcatcaagaaaa tagagctggg 4440 ccatgacggg gcctcccctg agagctgctg gctggtggaagagttgtgtt tggcagtgcc 4500 cacccagggc accaagtaca tgttgaactg taactgctggctggccaagg acagaggcga 4560 cggcatcacc tcccgtgtct tcgacctctt ggatgccatggtggtgaaca ttggggtgaa 4620 ggttctctat gaaatgacgg tgtggacagg ggatgtggttggcgggggca ctgactccaa 4680 catcttcatg accctctacg gcatcaacgg gagcacagaggagatgcagc tggacaaaaa 4740 gaaagccagg tttgagcggg agcagaacga caccttcatcatggagatcc tagacattgc 4800 tccattcacc aagatgcgga tccggattga tggcctgggcagtcggccgg agtggttcct 4860 ggagaggatc ctactgaaga acatgaacac tggagacctgaccatgttct actatggaga 4920 ctggctgtcc cagcggaagg gcaagaagac cctggtgtgtgaaatgtgtg ccgttatcga 4980 tgaggaagaa atgatggagt ggacctccta caccgtcgcagttaagacca gcgacatcct 5040 gggagcaggc actgatgcca acgtgttcat catcatcttcggggagaacg gggatagtgg 5100 gacactggcc ctgaagcagt cggcaaactg gaacaagtttgagcggaaca acacggacac 5160 attcaacttc cctgacatgc tgagcttggg ccacctctgcaagctgaggg tctggcacga 5220 caacaaaggg atatttcctg gctggcatct gagctatgtcgatgtgaagg acaactcccg 5280 cgacgagacc ttccacttcc agtgtgactg ctggctctccaagagtgagg gtgacgggca 5340 gacggtccgc gactttgcct gtgccaacaa caagatctgtgatgagctgg aagagaccac 5400 ctacgagatc gtcatagaaa cgggcaacgg aggcgaaaccagggagaacg tctggctcat 5460 cctggagggc aggaagaacc gatccaaaga gtttctcatggaaaattctt ctaggcagcg 5520 ggcctttagg aaggggacca cagacacgtt tgagtttgacagcatctact tgggggacat 5580 tgcctccctc tgtgtgggcc accttgccag ggaagaccggtttatcccca agagagaact 5640 tgcctggcat gtcaagacca tcaccatcac cgagatggagtacggcaatg tgtacttctt 5700 taactgtgac tgcctcatcc ccctcaagag gaagaggaagtacttcaagg tattcgaggt 5760 taccaagacg acagagagct ttgccagcaa ggtccagagcctggtgcccg tcaagtacga 5820 agtcatcgtg acaacaggct atgagccagg ggcaggcactgatgccaacg tcttcgtgac 5880 catctttggg gccaacggag acacaggcaa gcgggagctgaagcagaaaa tgcgcaacct 5940 cttcgagcgg ggcagcacag accgcttctt cctggagacgctggagctgg gtgagctgcg 6000 caaggtgcgc ctggagcacg acagcagtgg ctactgctcaggctggctgg tggagaaggt 6060 ggaggtcacc aacaccagca ccggcgtggc caccatcttcaactgtggca ggtggctgga 6120 caagaagcgg ggggatggac tcacctggag agacctcttcccttctgtct gaggggctag 6180 ggcccccacc ctctcactga gatgccccaa tctcacatttctgccctcca ccttggcggt 6240 cagcagccct tcaaagcctc tagcattggc actgggggctagcagtccac tgagaacttc 6300 atggggtcct gctccccacc ctaccc 6326 14 1561 DNAHomo sapiens misc_feature Incyte ID No 7474074CB1 14 gctcgaccagtactcagctt gactgacatt ttcttatttc agataataaa agaccatgcc 60 ttgaattctctcagctaagt gtaaaggatt ccttcagaga tttatttatt ccgagaatag 120 agaccattctgatgatgtat acaaggaaca acctaaactg tgctgagcca ctgtttgaac 180 aaaataactcacttaatgtt aatttcaaca cacaaaagaa aacagtctgg cttattcacg 240 gatacagaccagtaggctcc atcccattat ggcttcagaa cttcgtaagg attttgctga 300 atgaagaagatatgaatgta attgtagtag actggagccg gggtgctaca acttttattt 360 ataatagagcagttaaaaac accagaaaag ttgctgtgag tttgagtgtg cacattaaaa 420 atcttttgaagcatggtgca tctcttgaca attttcattt cataggtgtg agcttagggg 480 ctcatatcagtggatttgtt ggaaagatat ttcatggtca acttggaaga ataacaggtc 540 ttgaccctgctgggccaagg ttctccagaa aaccaccata tagcagatta gattacacgg 600 atgcaaagtttgtggatgtc atccattctg actccaatgg tttaggcatt caagagccct 660 tgggacatatagatttttat ccaaatggag gaaataaaca acctggctgt cctaaatcaa 720 ttttctcaggaattcaattc attaaatgca accaccagag agcagttcac ttgttcatgg 780 catctttagaaacaaactgc aattttattt catttccttg tcgttcatac aaagattaca 840 agactagcttatgtgtggac tgtgactgtt ttaaggaaaa atcatgtcct cggctgggtt 900 atcaagccaagctatttaaa ggtgttttaa aagaaaggat ggaaggaaga cctcttagga 960 ccactgtgtttttggataca agtggtacat atccattctg tacctattat tttgttctca 1020 gtataattgttccagataaa actatgatgg atggctcgtt ttcatttaaa ttattaaatc 1080 agcttgaaatgattgaagag ccaaggcttt atgaaaagaa caaaccattt tataaacttc 1140 aagaagtcaagattcttgct caattttata atgactttgt aaatatttca agcattggtt 1200 tgacatatttccagagctca aatctgcagt gttccacatg cacatacaag atccagagtc 1260 tcatgttaaaatcacttaca tacccaaaaa gaccaccact ttgcaggtat aatattgtac 1320 ttaaagaaagagaggaagtg tttcttaatc caaacacatg tacgccaaag aacacataag 1380 atgccttcttccatcaaatg cacttgcttg tgaattaatg gacttgtaaa tgaaacaatg 1440 caatcagtcttttataatac actgttcaat ttgagattca agtatttcta tttcttggaa 1500 aaaattttaagaatcaaaaa taaagaaaat aaaaagtgca tacagttaaa cattccaaaa 1560 a 1561 154941 DNA Homo sapiens misc_feature Incyte ID No 72024970CB1 15attccttggt ggccctggag ggtggatagg ctggcctggg ggccatcagg acagcaggtg 60acggtcaggc caatgccagc cgggcctggg cacagccctg tgggggcttc ggagggccct 120gaggaggagg aggaagaggc agaggagaga aggccccacg gaggtcctgt cgccagcgct 180gccactgcct gacctccgct gcccgaaggc cggtgggcct ctgtggcctc cgtgaagcag 240gcccggctgt cgtcaggcca tgtctggtcc atggccctcc cccgacagcc ggaccaaggg 300aacggtggcc tggctggcgg aggtactcct ctggttggag ggagtgtggt gctgtcttca 360gagtggcagc tcggccccct ggtggagcgg tgcatgggtg ccatgcaaga ggggatgcag 420atggtgaagc tgcgtggcgg ctccaagggc ctggtccgct tctactacct ggacgagcac 480cgctcctgca tccgctggag gccctcacgc aagaacgaga aggccaagat ctccatcgac 540tccatccagg aggtgagtga ggggcggcag tcggaggtct tccagcgcta ccctgacggc 600agcttcgacc ccaactgctg cttcagcatc taccacggca gccaccgcga gtcgctggac 660ctggtctcca ccagcagcga ggtggcgcgc acctgggtca ctggcctgcg ctacctcatg 720gccggcatca gcgacgagga cagcctggct cgccgccagc gcaccaggga ccagtggctg 780aagcagacgt ttgacgaggc cgacaagaac ggggatggca gcctgagcat tggcgaggtc 840ctgcagctgc tgcacaagct caacgtgaac ctgccccggc agagggtgaa gcagatgttc 900agggaagcgg acacggatga ccaccaaggg acgctgggtt ttgaagagtt ctgtgccttc 960tacaagatga tgtccacccg ccgggacctc tacctgctca tgctgaccta cagcaaccac 1020aaggaccacc tggatgccgc cagcctgcag cgcttcctgc aggtggagca gaagatggcg 1080ggtgtgaccc tcgagagctg ccaggacatc atcgagcagt ttgagccatg cccagaaaac 1140aagagtaagg ggctgctggg cattgatggc ttcaccaact acaccaggag ccctgctggt 1200gacatcttca accctgagca ccaccatgtg caccaggaca tgacgcagcc gctgagccac 1260tacttcatca cctcgtccca caacacctac ctcgtgggtg accagctcat gtcccagtca 1320cgggtggaca tgtatgcttg ggtcctgcag gctggctgcc gctgcgtgga ggtggactgc 1380tgggatgggc ccgacgggga gcccattgtg caccatggct acactctgac ttccaagatc 1440ctcttcaaag acgtcattga aaccatcaac aaatatgcct tcatcaagaa tgagtaccca 1500gtgatcctgt ccatcgaaaa ccactgcagt gtcatccagc agaagaaaat ggcccagtat 1560ctgactgaca tccttgggga caagctggac ctgtcatcag tgagcagtga agatgccacc 1620acactcccct ctccacagat gctcaagggc aagatcctcg tgaaggggaa gaagctccca 1680gccaacatca gcgaggatgc ggaggaaggc gaggtgtctg atgaggacag tgctgatgag 1740attgacgatg actgcaagct cctcaatggg gatgcatcca ccaatcgaaa gcgtgtagaa 1800aacactgcta agaggaaact ggattccctc atcaaagagt cgaagattcg ggactgtgag 1860gaccccaaca acttctccgt ctccacactg tccccatctg gaaagctcgg acgcaagagc 1920aaggctgaag aggacgtgga gtctggggag gatgccgggg ccagcagacg caatggccgc 1980ctcgtcgtgg gaagcttctc caggcgcaag aagaagggca gcaagctgaa gaaggcggcc 2040agcgtggagg agggagatga gggtcaggac tccccgggag gccagagccg aggggcgacc 2100cggcagaaga agaccatgaa gctgtcccgg gccctctctg acctggtgaa gtacaccaag 2160tccgtggcca cccacgacat agagatggag gcggcgtcca gctggcaggt gtcgtccttc 2220agcgagacca aggcccacca gattctgcag cagaagccgg cgcagtacct acgcttcaac 2280cagcagcagc tctcccgcat ctacccctcc tcctaccgtg tggactccag caactacaac 2340ccgcagccct tctggaacgc cggctgccaa atggttgccc tgaactacca gtcagagggg 2400cggatgctgc agctgaaccg agccaagttc agcgccaacg gtggctgcgg ctacgtactc 2460aagcctgggt gcatgtgcca gggcgtgttc aaccccaact cggaggaccc cctgcccggg 2520cagctcaaga agcagctggt gctccggatc atcagtggcc agcagcttcc caagccgcgc 2580gactccatgc tgggggaccg tggggagatc atcgacccct ttgtggaggt ggagatcatt 2640gggctccctg tggactgcag cagggagcag acccgcgtgg tggacgacaa cgggttcaac 2700cccacctggg aggagaccct ggttttcatg gtgcacatgc cggagatcgc gctggtccgc 2760ttcctcgtct gggaccacga tcccatcggg cgtgacttca ttggccagag gacgctggcc 2820ttcagcagca tgatgccagg ctacagacac gtgtacctag aagggatgga agaggcctcc 2880atcttcgtgc atgtggctgt cagtgacatc agcggtaagg tcaagcaggc tctgggccta 2940aaaggcctct tcctccgagg cccaaagccc ggctcgctgg acagtcatgc tgctgggcgg 3000cccccggccc ggccctccgt tagccagcgg atcctgcggc gcacggccag cgccccgacc 3060aagagccaga agccgggccg caggggcttc ccggagctgg tcctgggtac acgggacaca 3120ggctccaagg gggtggcaga cgatgtggtg ccccccgggc ccggacctgc tccggaagcc 3180ccagcccagg aggggcccgg cagcggcagc ccccgaggta aggcgccagc tgcggtggca 3240gagaagagcc ctgtgcgagt gcggcccccg cgtgtcctgg acggccccgg gcctgctggg 3300atggccgcca catgcatgaa gtgtgtggtg ggatcctgcg ccggcgtgaa caccgggggc 3360ccgcagaggg agcggccacc cagcccgggg cctgcaagca ggcaggcagc cattcgccag 3420cagccccggg cccgggctga ctcactgggg gccccctgct gtggcctgga ccctcacgct 3480atcccgggga gaagcagaga ggcccccaag ggtcctgggg cctggaggca gggtccaggc 3540ggtagcggct ccatgtcctc ggactccagc agcccagaca gcccgggcat ccccgaaagg 3600tccccccgct ggcctgaggg tgcctgcagg caaccggggg ccctgcaggg agagatgagt 3660gccttgtttg ctcaaaagct ggaggagatc aggagtaaat cccccatgtt ctccgccgtt 3720aggaactgag agcggcgagt gacagacacc cgccccctct ccacgcagcg gccactcccc 3780ccactgtgca gcctggaaac catcgctgag gagcccgccc caggccctgg tcccccgcca 3840ccagcggctg tccccaccag ctcttctcag ggacggcccc cataccccac aggacccgga 3900gccaatgtgg caagccccct agaggacact gaggagcccc gagacagcag gcctcggccg 3960tgcaacggcg agggcgccgg cggggcatac gagagggccc ccggcagcca gacggacggc 4020aggagccagc cccggaccct gggccacctg cccgtgatta gaagggtgaa gagtgagggg 4080caggtgccca cggagcccct gggagggtgg cggcccctgg ccgctccctt tccagctcct 4140gccgtgtact ccgatgccac gggcagtgac ccgctgtggc agcggctgga gccatgtggc 4200caccgagaca gcgtttcctc ctcctccagc atgtcatcca gcgacactgt cattgacctc 4260tccctgccca gcctgggcct gggccgcagc cgtgagaacc tcgctggagc ccacatggga 4320cgcctgcccc ccaggcccca ctcggcttcg gctgcccgcc cagacctgcc acctgtgacc 4380aagagcaaat ccaaccccaa ccttcgggct acaggccagc ggcctcccat acctgacgaa 4440ctgcagccca ggtccctggc cccaaggatg gctggcctcc ccttccggcc tccctggggc 4500tgcctttccc tggtgggcgt gcaggactgc cccgtggctg ccaagtccaa gagcctgggc 4560gacctcactg ctgatgactt tgcccctagc tttgagggcg gctcccgcag actgagccac 4620agcctgggcc tcccgggagg gacacggcgg gtgtcggggc cagggtgaga cgggacaccc 4680tgacagagca gctgcgctgg ctcactgtct tccagcaggc aggagacatc acgtcaccca 4740ccagcctggg cccggctggg gagggggtgg caggggccct ggttttgtgc ggcgctcctc 4800ctcccgcagc acagcgcgtg cgtgcattgc agcggccgca ggccggagcg cgcgaactga 4860ggctgggcgg gggacccgag gggggggccc cggggccgtt ctggccaggt gtgtttccgc 4920tcagggctgg ctcctttacc t 4941 16 4159 DNA Homo sapiens misc_featureIncyte ID No 6131380CB1 16 tctcggtctt cggtgcgaga tcactttgtt cctggagacagtgtacagag cttggaattc 60 tctgaggggt tcctgagatg gcgccattca agccagggggttgaatagct tgactcttca 120 tttcagcagc acgattgacc cctcagtgga tcagcagcgattcattccac acgtatttag 180 ggtccttggt gaattttgtc atggttattt aaggaaccttgcctagaagt cccaacttgc 240 agttccccat cgacgggaag gcttggactc caagatgattataaaggaat atcggattcc 300 tctgccaatg accgtggagg agtaccgcat cgcccagctgtacatgatac agaagaagag 360 ccgtaacgag acatatggcg aaggcagcgg cgtggagatcctggagaacc ggccgtacac 420 agatggccca ggcggctctg ggcagtacac acacaaggtgtatcatgtgg gcatgcacat 480 tcccagctgg ttccgctcca tcctgcccaa ggcagccctgcgggtggtgg aggagtcttg 540 gaatgcctac ccctacaccc gaaccaggtt cacctgtcctttcgtggaga aattctccat 600 cgacattgaa accttttata aaactgatgc tggagaaaaccccgacgtgt tcaacctctc 660 tcctgtggaa aagaaccagc tgacaatcga cttcatcgacattgtcaaag accctgtgcc 720 ccacaacgag tataagacag aagaggaccc caagctgttccagtcaacca agacccagcg 780 ggggcccctg tccgagaact ggatcgagga gtacaagaagcaggtcttcc ccatcatgtg 840 cgcatacaag ctctgcaagg tggagttccg ctactggggcatgcagtcca agatcgagag 900 gttcatccac gacaccggac tacggagggt gatggtgcgggctcaccggc aggcctggtg 960 ctggcaggac gagtggtatg ggctgagcat ggagaacatccgggagctgg agaaggaggc 1020 acagctcatg ctttcccgta agatggccca gttcaatgaggatggtgagg aggccactga 1080 gctcgtcaag cacgaagccg tctcggacca gacctctggggagcccccgg agcccagcag 1140 cagcaatggg gagcccctag tggggcgcgg cctcaagaaacagtggtcca catcctccaa 1200 gtcgtctcgg tcgtccaagc ggggagcgag tccttcccgccacagcatct cagagtggag 1260 gatgcagagt attgccaggg actcggatga gagctcagatgatgagttct tcgatgcgca 1320 cgaggacctg tccgacacag aggaaatgtt ccccaaggacatcaccaagt ggagctccaa 1380 tgacctcatg gacaagatcg agagcccaga gccggaagacacacaagatg gtctgtaccg 1440 ccagggtgcc cctgagttca gggtggcctc cagtgtggagcagctgaaca tcatagagga 1500 cgaggttagc cagccgctgg ctgcaccgcc ctccaagatccacgtgctgc tactggtgct 1560 gcacggaggc accatcctgg acacaggcgc cggggaccccagctccaaga agggcgatgc 1620 taacaccatc gccaacgtgt tcgacaccgt catgcgcgtgcactacccca gcgccctggg 1680 ccgccttgcc atccgcctgg tgccctgccc gcccgtctgctctgacgcct ttgccctggt 1740 ctccaacctc agcccctaca gccatgacga aggctgtctgtccagcagtc aggaccacat 1800 tcccctggct gccctccccc tgctggccac ctcctccccccagtaccagg aggcagttgc 1860 cacagtgatt cagcgagcca accttgccta tggggacttcatcaagtccc aggagggcat 1920 gaccttcaat gggcaggtct gcctgattgg ggactgcgtcgggggcatcc tggcatttga 1980 tgccctgtgc tacagtaacc agccggtgtc tgagagtcagagcagcagcc gccggggcag 2040 cgtggtcagc atgcaggaca atgacctgct gtccccgggcatcctgatga atgcagcaca 2100 ctgctgcggt ggtggcggtg gcggcggtgg cggtggtggcagcagtggtg gtggtggcag 2160 tagtggtggc tccagcctgg agagcagtcg gcacctgagccgaagcaacg tcgacatccc 2220 ccgcagcaac ggcactgagg accccaaaag gcaactgccccgcaagagga gcgactcatc 2280 cacctacgag ctggatacca tccagcagca ccaggccttcctgtccagcc tccatgccag 2340 cgtgctgagg actgagccct gctcacgcca ttccagcagctccaccatgc tggatggcac 2400 aggtgccctg ggcaggtttg actttgagat caccgacctcttcctcttcg ggtgcccgct 2460 ggggctggtc ctggccttga ggaagactgt catcccagccctggatgttt tccagctgcg 2520 gccggcctgc cagcaagtct acaacctctt ccaccccgcggacccgtcag cttcacgcct 2580 ggagccgctg ctggaacggc gctttcacgc cctgccgcctttcagcgtcc cccgctacca 2640 acgctacccg ctgggggatg gctgctccac gctgctggatgtgctccaga cccacaatgc 2700 agccttccaa gagcatggcg ccccctcctc gccgggcactgcccctgcca gtcgtggctt 2760 ccgccgagcc agtgagatca gcatcgccag ccaggtgtcaggcatggctg agagctacac 2820 ggcatccagc atcgcccagg tcgctgcaaa gtggtggggccagaagcgga tcgactacgc 2880 cctgtactgc cctgacgccc tcacggcctt ccccacggtggctctgcctc acctcttcca 2940 cgccagctac tgggagtcaa cagacgtggt ctcctttctgctgagacagg tcatgaggca 3000 tgacaactcc agcatcttgg agctggatgg caaggaagtgtcggtgttca ccccctcaaa 3060 gccaagggag aagtggcagc gcaagcggac ccacgtgaagctgcggaacg tgacggccaa 3120 ccaccggatc aatgatgccc ttgccaatga ggacggcccccaggttctga cgggcaggtt 3180 catgtatggg cccctggaca tggtcaccct gactggggagaaggtggatg tgcacatcat 3240 gacccagccg ccctcaggcg agtggctcta cctggatacgctggtgacca acaacagtgg 3300 gcgtgtctcc tacaccatcc ctgagtcgca ccgcctgggcgtgggtgtct accctatcaa 3360 gatggtggtc aggggagacc acacgtttgc cgacagctacatcaccgtgc tgcccaaggg 3420 cacagagttc gtggtcttca gcatcgacgg ttcctttgccgctagcgtgt ccatcatggg 3480 cagcgacccc aaggtgcggg ccggggccgt ggacgtggtgcggcactggc aggacctggg 3540 ctacctcatc atctacgtga cgggccggcc cgacatgcagaagcagcggg tggtggcgtg 3600 gctggcccag cacaacttcc cccatggcgt ggtgtccttctgtgacggcc tggtgcatga 3660 cccgctgcgg cacaaggcca acttcctgaa gctgctcatctccgagctgc acctgcgcgt 3720 gcacgcggcc tatggctcca ccaaggacgt ggcggtgtacagcgccatta gcctgtcccc 3780 catgcagatc tacatcgtgg gccggcccac caagaagctgcagcagcagt gccagttcat 3840 cacggatggc tacgcggccc acctggcgca gctgaagtacagccaccggg cgcggcccgc 3900 tcgcaacacg gccacccgca tggcgctgcg caagggcagcttcggcctgc ccggccaggg 3960 cgactttctg cgctcccgga accacctgct tcgcaccatctcggcccagc ccagcgggcc 4020 cagccaccgg cacgagcgga cacagagcca ggcggatggcgagcagcggg gccagcgcag 4080 catgagtgtg gcggccggct gctggggccg cgccatgactggccgcctgg agccgggggc 4140 agccgcgggc cccaagtag 4159 17 1481 DNA Homosapiens misc_feature Incyte ID No 643681CB1 17 atttgtgtaa tgtctttgtctccattagac ctttattatt tgatttacgt ctggtcttga 60 actcctgacc tcaggtggtccacccgcctc ggcctcccaa agtgctggca ttacaggcgt 120 gaaccaccgt gcctggccggaagtctttaa aaaataaagt gattctactc ttctaagctt 180 acagagacca gaccaggtgaatgtaactgg ggaaaatcaa gatggtacct ctctgcatta 240 tcccgccaga cactgtattttatgcattca tgtctaggat acagtgtgaa aattaaaaag 300 tttagagggc agatgcaattgtggcaagtg acctgccaat aaagcaggtg cagctataga 360 agctggcata ggtatatccttaatggtgct ttctccctgg gcttgtcttt ttgttgtttt 420 ttttccccta tattcagaagctccttgaga agtgataaac acctccagct ttctaacatc 480 ctccccacac catctcaccatatccatctc ccagcatcca tctgcattca gctaagggcg 540 ggaaactgac ctagtgcctgtgttgcagac catttctgag gtctccacca tccaaggagg 600 cacagccgtc attactgtcctccatgcctt cagcagcccc cctcacagct aaggtacata 660 ccaccccttc tgccgcgcctccacccctgg caccaaggtc ttctgctgct tatgtctaaa 720 gggatcacct atatttaactgcctcagtga cctaacctct ttcttctcat gtgccagatg 780 ttaagatgaa ggaggaatacaacacatact caagcctcag cctgtttagt tgttttcact 840 ggggctcgct tttctgggacggtatttatt atcagactgg caagcctaac tccataggtt 900 tacaggaagt agggatatttttataaaaca attgtgtcct ccccacattt tgctatgtta 960 atatttgctt ctaacaatttgcagctgttt cactttttcc tcatttgtct ctaagttgaa 1020 ggctttgttg gaggggacagagcacaggaa cagccttgac agtctgtaat tattgtacag 1080 atattttaat agcatataaataagtatatt ccttttattt tgaaacaaaa atgatcagac 1140 actgcctttt gtgtgtttgctgcctgtggc atcctttttt aaaaagactg ttacatatta 1200 aaatagtgta catatataaatattacctct tttgctgtac agttgtgata gagactgaag 1260 attttatttt ttgtgtgctttttataagaa aaaaattaat acactaaaga atcttgctga 1320 tgtgattgta atgtacctatgtaacttatt tacttttgaa tgttcttctg tatctttaaa 1380 ccttttatta aataaggttttaaaaattaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1440 aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa c 1481 18 1841 DNA Homo sapiens misc_featureIncyte ID No 6897474CB1 18 gctctgctca gttctctgtg cctgtctccc tccagcactgccgaggttct ctgccgaggc 60 caaccagaaa taccccttgg aagctggaat cctgcaacaatggcccaggg tgtcctctgg 120 atcctactcg gattgctact gtggtcagac ccagggacagcctccctgcc cctgctcatg 180 gactctgtca tccaggccct ggctgagctg gagcagaaagtgccagctgc caagaccaga 240 cacacagctt ctgcgtggct gatgtcagct ccaaactctggcccccacaa tcgcctctac 300 cacttcctgc tgggggcatg gagcctcaat gctacagagttggatccctg cccactaagc 360 ccagagctgt taggcctgac caaggaggtg gcccgacatgacgtacgaga agggaaggaa 420 tatggggtgg tgctggcacc tgatggctcg accgtggctgtggagcctct gctggcgggg 480 ctggaggcag ggctgcaagg gcgcagggtc ataaatttgcccttggacag catggctgcc 540 ccttgggaga ctggagatac ctttccagat gttgtggccattgctccaga tgtaagagcc 600 acctcctccc caggactcag ggatggctct ccagatgtcaccactgcaga tattggagcc 660 aacactccag atgctacaaa aggctgtcca gatgtccaagcttccttgcc agatgccaaa 720 gccaagtccc caccgaccat ggtggacagc ctcctggcagtcaccctggc tggaaacctg 780 ggcctgacct tcctccgagg ttcccagacc cagagccatccagacctggg aactgagggc 840 tgctgggacc agctctctgc ccctcggacc tttacgcttttggaccccaa ggcatctctg 900 ttaaccatgg ccttcctcaa tggcgccctg gatggggtcatccttggaga ctacctgagc 960 cggactcctg agccccggcc atccctcagc cacttgctgagccagtacta tggggctggg 1020 gtggccagag acccagggtt ccgcagcaac ttccgacggcagaacggtgc tgctctgact 1080 tcagcctcca tcctggccca gcaggtgtgg ggaacccttgtccttctaca gaggctggag 1140 ccagtacacc tccagcttca gtgcatgagc caagaacagctggcccaggt ggctgccaat 1200 gctaccaagg aattcactga ggccttcctg ggatgcccggccatccaccc ccgctgccgc 1260 tggggagcgg cgccttatcg gggccgcccg aagctgctgcagctgccgct gggattcttg 1320 tacgtgcatc acacctacgt gcctgcacca ccctgcacggacttcacgcg ctgcgcagcc 1380 aacatgcgct ccatgcagcg ctaccaccag gacacgcaaggctggggaga catcggctac 1440 agtttcgtgg tgggctcgga cggctacgtg tacgagggacgcggctggca ctgggtgggc 1500 gcccacacgc tcggccacaa ctcccggggc ttcggcgtggccatagtggg caactacacc 1560 gcggcgctgc ccaccgaggc cgctctgcgc acggtgcgcgacacgctccc gagttgtgcg 1620 gtgcgcgccg gcctcctgcg gccagactac gcgctgctgggccaccgcca gctggtgcgc 1680 accgactgcc ccggcgacgc gctcttcgac ctgctgcgcacctggccgca cttcaccgcg 1740 actgttaagc caagacctgc caggagtgtc tctaagagatccaggaggga gccaccccca 1800 aggaccctgc cagccacaga cctccaataa agacagcatg g1841

What is claimed is:
 1. An isolated polypeptide selected from the groupconsisting of: a) a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1-9, b) a polypeptidecomprising a naturally occurring amino acid sequence at least 90%identical to an amino acid sequence selected from the group consistingof SEQ ID NO:1, SEQ ID NO:5-6, and SEQ ID NO:9, c) a polypeptidecomprising a naturally occurring amino acid sequence at least 91%identical to an amino acid sequence selected from the group consistingof SEQ ID NO:2-4 and SEQ ID NO:7, d) a polypeptide comprising anaturally occurring amino acid sequence at least 99% identical to theamino acid sequence of SEQ ID NO:8, e) a biologically active fragment ofa polypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-9, and f) an immunogenic fragment of apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1-9.
 2. An isolated polypeptide of claim 1comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 1-9.
 3. An isolated polynucleotide encoding a polypeptide ofclaim
 1. 4. An isolated polynucleotide encoding a polypeptide of claim2.
 5. An isolated polynucleotide of claim 4 comprising a polynucleotidesequence selected from the group consisting of SEQ ID NO:10-18.
 6. Arecombinant polynucleotide comprising a promoter sequence operablylinked to a polynucleotide of claim
 3. 7. A cell transformed with arecombinant polynucleotide of claim
 6. 8. A transgenic organismcomprising a recombinant polynucleotide of claim
 6. 9. A method ofproducing a polypeptide of claim 1, the method comprising: a) culturinga cell under conditions suitable for expression of the polypeptide,wherein said cell is transformed with a recombinant polynucleotide, andsaid recombinant polynucleotide comprises a promoter sequence operablylinked to a polynucleotide encoding the polypeptide of claim 1, and b)recovering the polypeptide so expressed.
 10. A method of claim 9,wherein the polypeptide comprises an amino acid sequence selected fromthe group consisting of SEQ ID NO:1-9.
 11. An isolated antibody whichspecifically binds to a polypeptide of claim
 1. 12. An isolatedpolynucleotide selected from the group consisting of: a) apolynucleotide comprising a polynucleotide sequence selected from thegroup consisting of SEQ ID NO:10-18, b) a polynucleotide comprising anaturally occurring polynucleotide sequence at least 90% identical to apolynucleotide sequence selected from the group consisting of SEQ IDNO:10-16 and SEQ ID NO:18, c) a polynucleotide comprising a naturallyoccurring polynucleotide sequence at least 91% identical to thepolynucleotide sequence of SEQ ID NO:17, d) a polynucleotidecomplementary to a polynucleotide of a), e) a polynucleotidecomplementary to a polynucleotide of b), f) a polynucleotidecomplementary to a polynucleotide of c), and g) an RNA equivalent ofa)-f).
 13. An isolated polynucleotide comprising at least 60 contiguousnucleotides of a polynucleotide of claim
 12. 14. A method of detecting atarget polynucleotide in a sample, said target polynucleotide having asequence of a polynucleotide of claim 12, the method comprising: a)hybridizing the sample with a probe comprising at least 20 contiguousnucleotides comprising a sequence complementary to said targetpolynucleotide in the sample, and which probe specifically hybridizes tosaid target polynucleotide, under conditions whereby a hybridizationcomplex is formed between said probe and said target polynucleotide orfragments thereof, and b) detecting the presence or absence of saidhybridization complex, and, optionally, if present, the amount thereof.15. A method of claim 14, wherein the probe comprises at least 60contiguous nucleotides.
 16. A method of detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide of claim 12, the method comprising: a) amplifyingsaid target polynucleotide or fragment thereof using polymerase chainreaction amplification, and b) detecting the presence or absence of saidamplified target polynucleotide or fragment thereof, and, optionally, ifpresent, the amount thereof.
 17. A composition comprising a polypeptideof claim 1 and a pharmaceutically acceptable excipient.
 18. Acomposition of claim 17, wherein the polypeptide comprises an amino acidsequence selected from the group consisting of SEQ ID NO:1-9.
 19. Amethod for treating a disease or condition associated with decreasedexpression of functional LIPAM, comprising administering to a patient inneed of such treatment the composition of claim
 17. 20. A method ofscreening a compound for effectiveness as an agonist of a polypeptide ofclaim 1, the method comprising: a) exposing a sample comprising apolypeptide of claim 1 to a compound, and b) detecting agonist activityin the sample.
 21. A composition comprising an agonist compoundidentified by a method of claim 20 and a pharmaceutically acceptableexcipient.
 22. A method for treating a disease or condition associatedwith decreased expression of functional LIPAM, comprising administeringto a patient in need of such treatment a composition of claim
 21. 23. Amethod of screening a compound for effectiveness as an antagonist of apolypeptide of claim 1, the method comprising: a) exposing a samplecomprising a polypeptide of claim 1 to a compound, and b) detectingantagonist activity in the sample.
 24. A composition comprising anantagonist compound identified by a method of claim 23 and apharmaceutically acceptable excipient.
 25. A method for treating adisease or condition associated with overexpression of functional LIPAM,comprising administering to a patient in need of such treatment acomposition of claim
 24. 26. A method of screening for a compound thatspecifically binds to the polypeptide of claim 1, the method comprising:a) combining the polypeptide of claim 1 with at least one test compoundunder suitable conditions, and b) detecting binding of the polypeptideof claim 1 to the test compound, thereby identifying a compound thatspecifically binds to the polypeptide of claim
 1. 27. A method ofscreening for a compound that modulates the activity of the polypeptideof claim 1, the method comprising: a) combining the polypeptide of claim1 with at least one test compound under conditions permissive for theactivity of the polypeptide of claim 1, b) assessing the activity of thepolypeptide of claim 1 in the presence of the test compound, and c)comparing the activity of the polypeptide of claim 1 in the presence ofthe test compound with the activity of the polypeptide of claim 1 in theabsence of the test compound, wherein a change in the activity of thepolypeptide of claim 1 in the presence of the test compound isindicative of a compound that modulates the activity of the polypeptideof claim
 1. 28. A method of screening a compound for effectiveness inaltering expression of a target polynucleotide, wherein said targetpolynucleotide comprises a sequence of claim 5, the method comprising:a) exposing a sample comprising the target polynucleotide to a compound,under conditions suitable for the expression of the targetpolynucleotide, b) detecting altered expression of the targetpolynucleotide, and c) comparing the expression of the targetpolynucleotide in the presence of varying amounts of the compound and inthe absence of the compound.
 29. A method of assessing toxicity of atest compound, the method comprising: a) treating a biological samplecontaining nucleic acids with the test compound, b) hybridizing thenucleic acids of the treated biological sample with a probe comprisingat least 20 contiguous nucleotides of a polynucleotide of claim 12 underconditions whereby a specific hybridization complex is formed betweensaid probe and a target polynucleotide in the biological sample, saidtarget polynucleotide comprising a polynucleotide sequence of apolynucleotide of claim 12 or fragment thereof, c) quantifying theamount of hybridization complex, and d) comparing the amount ofhybridization complex in the treated biological sample with the amountof hybridization complex in an untreated biological sample, wherein adifference in the amount of hybridization complex in the treatedbiological sample is indicative of toxicity of the test compound.
 30. Adiagnostic test for a condition or disease associated with theexpression of LIPAM in a biological sample, the method comprising: a)combining the biological sample with an antibody of claim 11, underconditions suitable for the antibody to bind the polypeptide and form anantibody:polypeptide complex, and b) detecting the complex, wherein thepresence of the complex correlates with the presence of the polypeptidein the biological sample.
 31. The antibody of claim 11, wherein theantibody is: a) a chimeric antibody, b) a single chain antibody, c) aFab fragment, d) a F(ab′)₂ fragment, or e) a humanized antibody.
 32. Acomposition comprising an antibody of claim 11 and an acceptableexcipient.
 33. A method of diagnosing a condition or disease associatedwith the expression of LIPAM in a subject, comprising administering tosaid subject an effective amount of the composition of claim
 32. 34. Acomposition of claim 32, wherein the antibody is labeled.
 35. A methodof diagnosing a condition or disease associated with the expression ofLIPAM in a subject, comprising administering to said subject aneffective amount of the composition of claim
 34. 36. A method ofpreparing a polyclonal antibody with the specificity of the antibody ofclaim 11, the method comprising: a) immunizing an animal with apolypeptide consisting of an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-9, or an immunogenic fragment thereof, underconditions to elicit an antibody response, b) isolating antibodies fromsaid animal, and c) screening the isolated antibodies with thepolypeptide, thereby identifying a polyclonal antibody which bindsspecifically to a polypeptide comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO:1-9.
 37. A polyclonal antibodyproduced by a method of claim
 36. 38. A composition comprising thepolyclonal antibody of claim 37 and a suitable carrier.
 39. A method ofmaking a monoclonal antibody with the specificity of the antibody ofclaim 11, the method comprising: a) immunizing an animal with apolypeptide consisting of an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-9, or an immunogenic fragment thereof, underconditions to elicit an antibody response, b) isolating antibodyproducing cells from the animal, c) fusing the antibody producing cellswith immortalized cells to form monoclonal antibody-producing hybridomacells, d) culturing the hybridoma cells, and e) isolating from theculture monoclonal antibody which binds specifically to a polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:1-9.
 40. A monoclonal antibody produced by a method of claim39.
 41. A composition comprising the monoclonal antibody of claim 40 anda suitable carrier.
 42. The antibody of claim 11, wherein the antibodyis produced by screening a Fab expression library.
 43. The antibody ofclaim 11, wherein the antibody is produced by screening a recombinantimmunoglobulin library.
 44. A method of detecting a polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:1-9 in a sample, the method comprising: a) incubating theantibody of claim 11 with a sample under conditions to allow specificbinding of the antibody and the polypeptide, and b) detecting specificbinding, wherein specific binding indicates the presence of apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-9 in the sample.
 45. A method of purifying apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-9 from a sample, the method comprising: a)incubating the antibody of claim 11 with a sample under conditions toallow specific binding of the antibody and the polypeptide, and b)separating the antibody from the sample and obtaining the purifiedpolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-9.
 46. A microarray wherein at least oneelement of the microarray is a polynucleotide of claim
 13. 47. A methodof generating an expression profile of a sample which containspolynucleotides, the method comprising: a) labeling the polynucleotidesof the sample, b) contacting the elements of the microarray of claim 46with the labeled polynucleotides of the sample under conditions suitablefor the formation of a hybridization complex, and c) quantifying theexpression of the polynucleotides in the sample.
 48. An array comprisingdifferent nucleotide molecules affixed in distinct physical locations ona solid substrate, wherein at least one of said nucleotide moleculescomprises a first oligonucleotide or polynucleotide sequencespecifically hybridizable with at least 30 contiguous nucleotides of atarget polynucleotide, and wherein said target polynucleotide is apolynucleotide of claim
 12. 49. An array of claim 48, wherein said firstoligonucleotide or polynucleotide sequence is completely complementaryto at least 30 contiguous nucleotides of said target polynucleotide. 50.An array of claim 48, wherein said first oligonucleotide orpolynucleotide sequence is completely complementary to at least 60contiguous nucleotides of said target polynucleotide.
 51. An array ofclaim 48, wherein said first oligonucleotide or polynucleotide sequenceis completely complementary to said target polynucleotide.
 52. An arrayof claim 48, which is a microarray.
 53. An array of claim 48, furthercomprising said target polynucleotide hybridized to a nucleotidemolecule comprising said first oligonucleotide or polynucleotidesequence.
 54. An array of claim 48, wherein a linker joins at least oneof said nucleotide molecules to said solid substrate.
 55. An array ofclaim 48, wherein each distinct physical location on the substratecontains multiple nucleotide molecules, and the multiple nucleotidemolecules at any single distinct physical location have the samesequence, and each distinct physical location on the substrate containsnucleotide molecules having a sequence which differs from the sequenceof nucleotide molecules at another distinct physical location on thesubstrate.
 56. A polypeptide of claim 1, comprising the amino acidsequence of SEQ ID NO:1.
 57. A polypeptide of claim 1, comprising theamino acid sequence of SEQ ID NO:2.
 58. A polypeptide of claim 1,comprising the amino acid sequence of SEQ ID NO:3.
 59. A polypeptide ofclaim 1, comprising the amino acid sequence of SEQ ID NO:4.
 60. Apolypeptide of claim 1, comprising the amino acid sequence of SEQ IDNO:5.
 61. A polypeptide of claim 1, comprising the amino acid sequenceof SEQ ID NO:6.
 62. A polypeptide of claim 1, comprising the amino acidsequence of SEQ ID NO:7.
 63. A polypeptide of claim 1, comprising theamino acid sequence of SEQ ID NO:8.
 64. A polypeptide of claim 1,comprising the amino acid sequence of SEQ ID NO:9.
 65. A polynucleotideof claim 12, comprising the polynucleotide sequence of SEQ ID NO:10. 66.A polynucleotide of claim 12, comprising the polynucleotide sequence ofSEQ ID NO:11.
 67. A polynucleotide of claim 12, comprising thepolynucleotide sequence of SEQ ID NO:12.
 68. A polynucleotide of claim12, comprising the polynucleotide sequence of SEQ ID NO:13.
 69. Apolynucleotide of claim 12, comprising the polynucleotide sequence ofSEQ ID NO:14.
 70. A polynucleotide of claim 12, comprising thepolynucleotide sequence of SEQ ID NO:15.
 71. A polynucleotide of claim12, comprising the polynucleotide sequence of SEQ ID NO:16.
 72. Apolynucleotide of claim 12, comprising the polynucleotide sequence ofSEQ ID NO:17.
 73. A polynucleotide of claim 12, comprising thepolynucleotide sequence of SEQ ID NO:18.